Microdevices having a preferential axis of magnetization and uses thereof

ABSTRACT

This invention relates generally to the field of moiety or molecule isolation, detection and manipulation and library synthesis. In particular, the invention provides a microdevice, which microdevice comprises: a) a magnetizable substance; and b) a photorecognizable coding pattern, wherein said microdevice has a preferential axis of magnetization. Systems and methods for isolating, detecting and manipulating moieties and synthesizing libraries using the microdevices are also provided.

The present application is a continuation of U.S. application Ser. No.11/230,411, filed on Sep. 20, 2005, now U.S. Pat. No. 7,262,016, whichis a divisional of U.S. application Ser. No. 10/104,571, filed on Mar.21, 2002, now U.S. Pat. No. 7,015,047, which is a continuation-in-partof U.S. patent application Ser. No. 09/924,428, filed Aug. 7, 2001, nowpending. The content of the above U.S. patent application Ser. No.09/924,428 is incorporated by reference herein in its entirety.

TECHNICAL FIELD

This invention relates generally to the field of moiety or moleculeisolation, detection, manipulation and synthesis. In particular, theinvention provides a microdevice, which microdevice comprises: a) amagnetizable substance; and b) a photorecognizable coding pattern,wherein said microdevice has a preferential axis of magnetization.Systems and methods for isolating, detecting, manipulating andsynthesizing moieties using the microdevices are also provided.

BACKGROUND ART

High-density, high throughput biological and biochemical assays havebecome essential tools for diagnostic and research applications,particularly in areas involving the acquisition and analysis of geneticinformation. These assays typically involve the use of solid substrates.Examples of typical quantitative assays performed on solid substratesinclude measurement of an antigen by ELISA or the determination of mRNAlevels by hybridization. Solid substrates can take any form thoughtypically they fall into two categories—those using spherical beads orthose using planar arrays.

Planar objects such as slide- or chip-based arrays offer the advantageof allowing capture molecules, e.g., antibody or cDNA, of known identityto be bound at spatially distinct positions. Surfaces are easily washedto remove unbound material. A single mixture of analytes can be capturedon a surface and detected using a common marker, e.g., fluorescent dye.The identification of captured analytes is governed by the spatialposition of the bound capture molecule. Archival storage of the array isgenerally possible. Because the array corresponds to a stationary flatsurface, detection devices are generally simpler in design and havelower cost of manufacture than bead reading devices. One of thedifficulties of the planar array approach is the initial positioning ofthe capture molecule onto the surface. Techniques such as roboticdeposition (e.g., “Quantitative monitoring of gene expression patternswith a complementary DNA microarray” by Schena et al. Science,270:467-470 (1995)), photolithography (e.g., “Light-directed, spatiallyaddressable parallel chemical synthesis” by Fodor et. al. Science,251:767-773 (1991)), or ink-jet technologies (e.g., “High-densityoligonucleotide arrays” by Blanchard et al. Biosensors Bioelectronics,6/7:687-690 (1996)) are generally used. These methods have a number oflimitations. They require expensive instrumentation to generate highdensity arrays (greater than 1000 features/cm²), and there is no abilityto alter the pattern after manufacture, e.g., replace one capture cDNAwith another, consequently any alterations require a new manufacturingprocess and greatly increase expenses. Moreover, molecules bound tolarge flat surfaces exhibit less favorable reaction kinetics than domolecules that are free in solution.

One way around many of these problems is to use surfaces of smallparticles. Spherical beads have been the small particles of choicebecause of their uniform symmetry and their minimal self-interactingsurface. Small particles, however, suffer from the problem of beingdifficult to distinguish, e.g., a mixture of beads is not spatiallydistinct. A number of technologies have been developed to overcome thisproblem by encoding beads to make them distinguishable. Companies suchas the Luminex Corporation have developed methods of doing this byincorporating different mixtures of fluorescent dyes into beads to makethem optically distinguishable. In a similar manner, other researchershave developed ways of incorporating other optically distinguishablematerials into beads (e.g., “Quantum-dot-tagged microbeads formultiplexed optical coding of biomolecules” by Han et al. NatureBiotechnology, 19:631-635 (2001)). Furthermore, quantum dots, nanometerscale particles that are neither small molecules nor bulk solids, havealso been used for bead identification. Their composition and small size(a few hundred to a few thousand atoms) give these dots extraordinaryoptical properties that can be readily customized by changing the sizeor composition of the dots. Quantum dots absorb light, then quicklyre-emit the light but in a different color. The most important propertyis that the color of quantum dots—both in absorption and emission—can be“tuned” to any chosen wavelength by simply changing their size. GeniconSciences Corporation (Their “RLS” particles are of nano-sizes and havecertain “resonance light scattering (RLS) properties) also developedmicro-beads or nano-beads with optically distinguishable properties.However, in using any of these approaches, it is difficult tomanufacture more than 1,000 or so different encoded beads.

Beads are also the format of choice in combinatorial chemistry. Usingthe one-bead/one-compound procedure (also known as the split and mixprocedure) (see “The “one-bead-one-compound” combinatorial librarymethod” by Lam et al. Chem. Rev., 97:411-448 (1997)), it is possible togenerate huge libraries containing in excess of 10⁸ different molecules.However, the beads are not distinguishable in any way other than byidentifying the compound on a particular bead. Labeled “tea bags” whichcontain groups of beads displaying the same compound have been used todistinguish beads. Recently, IRORI has extended the tea bag technologyto small canisters containing either a radiofrequency transponder or anoptically encoded surface. This technology is generally limited toconstructing libraries on the order of 10,000 compounds, a singlecanister occupies ˜0.25 mL. Moreover, the technology is not well suitedto high-throughput-screening. PharmaSeq, Inc. uses individual substratescontaining transponders. These devices are 250μ×250μ×100μ. Largerlibraries can be synthesized directly onto a surface to form planararrays using photolithographic methods (such as those used byAffymetrix). However, such techniques have largely been restricted toshort oligonucleotides due to cost considerations and the lowerrepetitive yields associated with photochemical synthesis procedures(see e.g., “The efficiency of light-directed synthesis of DNA arrays onglass substrates” by Mc Gall et al. J. Am. Chem. Soc., 119:5081-5090(1997)). In addition, the available number of photo-labile protectinggroups is severely limited compared to the tremendous breadth anddiversity of chemically labile protecting groups that have beendeveloped over the past 30+ years for use on beads. Recently, SmartBeadsTechnologies has introduced microfabricated particles (e.g., stripparticles having dimensions of 100μ×10μ×1μ) containing bar codes thatcan be decoded using a flow-based reader. Microfabricated particles havethe advantage that a nearly infinite number of encoding patterns can beeasily incorporated into them. The difficulty lies in being able toeasily analyze mixtures of encoded particles. Since such particles tendto be flat objects as opposed to spherical beads, they tend to be moreprone to aggregation or overlapping as well as being more difficult todisperse.

Nicewarner-Pena et al., Science, 294(5540):137-41 (2001) recentlyreported synthesis of multimetal microrods intrinsically encoded withsubmicrometer stripes. According to Nicewarner-Pena et al., complexstriping patterns are readily prepared by sequential electrochemicaldeposition of metal ions into templates with uniformly sized pores. Thedifferential reflectivity of adjacent stripes enables identification ofthe striping patterns by conventional light microscopy. This readoutmechanism does not interfere with the use of fluorescence for detectionof analytes bound to particles by affinity capture, as demonstrated byDNA and protein bioassays.

A system incorporating the advantages of planar arrays and of encodedmicro-particles would address many of the problems inherent in theexisting approaches. Illumina, Inc. has attempted to do this byproviding a method of generating arrays of microbeads using etched glassfibers (e.g., “High-density fiber-optic DNA random microsphere array” byFerguson et al. Anal. Chem., 72:5618-5624 (2000)). However, Illumina'soligonucleotide based fluorescent-encoding microbeads are also limitedin the number of unique representations. BioArray Solutions has usedLight-controlled Electrokinetic Assembly of Particles near Surfaces(LEAPS) to form arrays of beads on surfaces (WO 97/40385). However, theLEAPS approach is still subject to the same restrictions as bead-basedtechniques with respect to the types of available encoding.

There exists needs in the art for microdevices and methods that can takethe advantages of both microfabricated particles and spatially distinctarrays. This invention address these and other related needs in the art.

DISCLOSURE OF THE INVENTION

In one aspect, the present invention is directed to a microdevice, whichmicrodevice comprises: a) a magnetizable substance; and b) aphotorecognizable coding pattern, wherein said microdevice has apreferential axis of magnetization. In a specific embodiment, thepresent microdevice does not comprise Pt, Pd, Ni, Co, Ag, Cu or Au forencoding purposes. In another specific embodiment, the presentmicrodevice does not comprise Pt, Pd, Ni, Co, Ag, Cu or Au.

In another aspect, the present invention is directed to a system forforming a microdevice array, which system comprises: a) a plurality ofthe microdevices, each of the microdevices comprising a magnetizablesubstance and a photorecognizable coding pattern, wherein saidmicrodevices have a preferential axis of magnetization; and b) amicrochannel array comprising a plurality of microchannels, saidmicrochannels are sufficiently wide to permit rotation of saidmicrodevices within said microchannels but sufficiently narrow toprevent said microdevices from forming a chain when the major axis ofsaid microdevices is substantially perpendicular to the major axis ofsaid microchannels when the said microdevices are subjected to anapplied magnetic field. In a specific embodiment, the microdevice usedin the present system does not comprise Pt, Pd, Ni, Co, Ag, Cu or Au forencoding purposes. In another specific embodiment, the microdevice usedin the present system does not comprise Pt, Pd, Ni, Co, Ag, Cu or Au.

In still another aspect, the present invention is directed to a methodfor forming a microdevice array, which method comprises: a) providing aplurality of the microdevices, each of the microdevices comprising amagnetizable substance and a photorecognizable coding pattern, whereinsaid microdevices have a preferential axis of magnetization; b)providing a microchannel array comprising a plurality of microchannels,said microchannels are sufficiently wide to permit rotation of saidmicrodevices within said microchannels but sufficiently narrow toprevent said microdevices from forming a chain when the major axis ofsaid microdevices is substantially perpendicular to the major axis ofsaid microchannels when the said microdevices are subjected to anapplied magnetic field; c) introducing said plurality of microdevicesinto said plurality of microchannels; and d) rotating said microdeviceswithin said microchannels by a magnetic force, whereby the combinedeffect of said magnetic force and said preferential axis ofmagnetization of said microdevices substantially separates saidmicrodevices from each other. In a specific embodiment, the microdeviceused in the present method does not comprise Pt, Pd, Ni, Co, Ag, Cu orAu for encoding purposes. In another specific embodiment, themicrodevice used in the present system does not comprise Pt, Pd, Ni, Co,Ag, Cu or Au.

In yet another aspect, the present invention is directed to a method forforming a microdevice array, which method comprises: a) providing aplurality of the microdevices, each of the microdevices comprising amagnetizable substance and a photorecognizable coding pattern, whereinsaid microdevices have a preferential axis of magnetization, on asurface suitable for rotation of said microdevices; and b) rotating saidmicrodevices on said surface by a magnetic force, whereby the combinedeffect of said magnetic force and said preferential axis ofmagnetization of said microdevices substantially separates saidmicrodevices from each other. In a specific embodiment, the microdeviceused in the present method does not comprise Pt, Pd, Ni, Co, Ag, Cu orAu for encoding purposes. In another specific embodiment, themicrodevice used in the present system does not comprise Pt, Pd, Ni, Co,Ag, Cu or Au.

In yet another aspect, the present invention is directed to a method forsynthesizing a library, which method comprises: a) providing a pluralityof microdevices, each of said microdevices comprises a magnetizablesubstance and a photorecognizable coding pattern, wherein saidmicrodevices have a preferential axis of magnetization and wherein saidphotorecognizable coding pattern corresponds to an entity to besynthesized on said microdevice; and b) synthesizing said entities onsaid microdevices, wherein said microdevices are sorted after eachsynthesis cycle according to said photorecognizable coding patterns,whereby a library is synthesized, wherein each of said microdevicescontains an entity that corresponds to a photorecognizable codingpattern on said microdevice and the sum of said microdevicescollectively contains a plurality of entities that is predeterminedbefore the library synthesis. In a specific embodiment, the microdeviceused in the present method does not comprise Pt, Pd, Ni, Co, Ag, Cu orAu for encoding purposes. In another specific embodiment, themicrodevice used in the present system does not comprise Pt, Pd, Ni, Co,Ag, Cu or Au. A library that is synthesized according to the abovemethod is also provided.

BRIEF DESCRIPTION OF THE DRAWINGS

FIG. 1 illustrates an example of a microdevice (MicroDisk) that isrectangular and consists of four regions. Magnetic bars are shown inlight gray. Dark gray region (e.g., made of the material Aluminum, Al)is an encoding region. The surrounding white edge (e.g. made of SiO₂)indicates the regions that encapsulate the magnetic bars and encodingregion. Arrow indicates direction of the external magnetic field. Thesedifferent regions are also located separately along the thicknessdirection. The magnetic bars and the encoding region are located in themiddle, and are encapsulated by the top and bottom layers thatcorrespond to the surrounding white edge. In an exemplary microdevice,the MicroDisk contains magnetic bars comprising soft magnetic material,e.g., CoTaZr or NiFe and is 90μ long by 70μ wide by 3.2μ thick.

FIG. 2 illustrates examples of possible arrangements of multipleMicroDisks constrained to a surface in the presence of a magnetic fieldwhose direction is indicated by the arrow.

FIG. 3 illustrates a short chain of MicroDisks constrained to a surfaceand further constrained in a channel while in the presence of a magneticfield whose direction is indicated by the arrow.

FIG. 4 illustrates the same short chain of MicroDisks shown in FIG. 3after the external magnetic field has been rotated by 90 degrees asindicated by the arrow.

FIG. 5 shows examples of MicroDisks containing different types ofmagnetic bars.

FIG. 6 shows examples of two types of encoding patterns: 2D datamatrixon the left and four character optical character recognition (OCR) onthe right.

FIG. 7 shows an exemplary microchannel device containing a loadingregion, guiding posts, microchannels, collection areas and fluidicconnections.

FIG. 8 shows 4 exemplary types of MicroDisks. Images show MicroDisksafter fabrication but before release from the wafer. Magnification is˜400×. A—Pair of rectangular magnetic bars, 2D bar code; B—Pair ofrectangular magnetic bars with tapered ends, 3-character OCR code;C—Pair of rectangular magnetic bars with “three-fingered” ends; 1D barcode; D—Five rectangular magnetic bars, 4-character OCR code.

FIG. 9 shows MicroDisks forming linear chains on a glass surface in thepresence of a magnetic field whose direction is indicated by the arrow.The 2D bar codes are fully exposed in this chain. Illumination is frombelow. Magnification is ˜400×.

FIG. 10 shows MicroDisks forming chains with some branching on a glasssurface in the presence of a magnetic field whose direction is indicatedby the arrow. Illumination is from below. Magnification is 400×.

FIG. 11 shows MicroDisks constrained to a 130μ/wide channel respondingto a magnetic field whose direction is indicated by the arrow. In theupper panel (A), the MicroDisks form a compact chain. Ninety (90)-degreerotation of the magnetic field as shown in the lower panel (B) resultsin the disks fully separating from each other. Illumination is fromabove. Magnification is ˜160×.

MODES OF CARRYING OUT THE INVENTION

For clarity of disclosure, and not by way of limitation, the detaileddescription of the invention is divided into the subsections thatfollow.

A. Definitions

Unless defined otherwise, all technical and scientific terms used hereinhave the same meaning as is commonly understood by one of ordinary skillin the art to which this invention belongs. All patents, applications,published applications and other publications referred to herein areincorporated by reference in their entirety. If a definition set forthin this section is contrary to or otherwise inconsistent with adefinition set forth in the patents, applications, publishedapplications and other publications that are herein incorporated byreference, the definition set forth in this section prevails over thedefinition that is incorporated herein by reference.

As used herein, “a” or “an” means “at least one” or “one or more.”

As used herein, “magnetic substance” refers to any substance that hasthe properties of a magnet, pertaining to a magnet or to magnetism,producing, caused by, or operating by means of, magnetism.

As used herein, “magnetizable substance” refers to any substance thathas the property of being interacted with the field of a magnet, andhence, when suspended or placed freely in a magnetic field, of inducingmagnetization and producing a magnetic moment. Examples of magnetizablesubstance include, but are not limited to, paramagnetic, ferromagneticand ferrimagnetic substances.

As used herein, “paramagnetic substance” refers to the substances wherethe individual atoms, ions or molecules possess a permanent magneticdipole moment. In the absence of an external magnetic field, the atomicdipoles point in random directions and there is no resultantmagnetization of the substances as a whole in any direction. This randomorientation is the result of thermal agitation within the substance.When an external magnetic field is applied, the atomic dipoles tend toorient themselves parallel to the field, since this is the state oflower energy than antiparallel position. This gives a net magnetizationparallel to the field and a positive contribution to the susceptibility.Further details on “paramagnetic substance” or “paramagnetism” can befound in various literatures, e.g., at Page 169-page 171, Chapter 6, in“Electricity and Magnetism” by B. I Bleaney and B. Bleaney, Oxford,1975.

As used herein, “ferromagnetic substance” refers to the substances thatare distinguished by very large (positive) values of susceptibility, andare dependent on the applied magnetic field strength. In addition,ferromagnetic substances may possess a magnetic moment even in theabsence of the applied magnetic field, and the retention ofmagnetization in zero field is known as “remanence”. Further details on“ferromagnetic substance” or “ferromagnetism” can be found in variousliteratures, e.g., at Page 171-page 174, Chapter 6, in “Electricity andMagnetism” by B. I Bleaney and B. Bleaney, Oxford, 1975.

As used herein, “ferrimagnetic substance” refers to the substances thatshow spontaneous magnetization, remanence, and other properties similarto ordinary ferromagnetic materials, but the spontaneous moment does notcorrespond to the value expected for full parallel alignment of the(magnetic) dipoles in the substance. Further details on “ferrimagneticsubstance” or “ferrimagnetism” can be found in various literatures,e.g., at Page 519-524, Chapter 16, in “Electricity and Magnetism” by B.I Bleaney and B. Bleaney, Oxford, 1975.

As used herein, “a photorecognizable coding pattern” refers to anycoding pattern that can be detected and/or assessed by photoanalysis(optical analysis). Any photorecognizable property can be used as thecharacteristics of the coding pattern. For example, thephotorecognizable coding pattern can be the material composition of themicrodevice or substrate itself, structural configuration of themicrodevice (e.g., a hole in the microdevice or the substrate or asubstance immobilized on the microdevice or the substrate), saidsubstance having an optical refractive property that is different fromthe optical refractive property of the microdevice or the substrate. Theversatility of the photorecognizable coding pattern can be based on theshape, number, position distribution, optical refractive property,material composition, or a combination thereof, of the microdevice orthe substrate, the hole(s), or other structural configurations, orcertain substance(s) located, deposited or immobilized on themicrodevice or the substrate. To facilitate optical analysis (orphotoanalysis) of encoding patterns, certain microdevices mayincorporate “orientation” marks or alignment markers. The orientationmarkers can be used for indicating which major surface is up and forhelping decode the patterns. 1-D and/or 2-D bar coding patterns can alsobe used as photorecognizable coding pattern in the present microdevices.

As used herein, “a photorecognizable coding pattern on said substrate”means that the photorecognizable coding pattern is located on, in, orwithin (or inside) the substrate so that the photorecognizable codingpattern is optically detectable. For example, the photorecognizablecoding pattern can be located on the surface or on top of the substrate.The photorecognizable coding pattern can also be located within orinside the substrate. In other embodiments, the substrate may havemultiple layers and the photorecognizable coding pattern can be locatedon the surface layer, on top of the surface layer, or can be locatedwithin or inside one or more layers.

As used herein, “the photorecognizable coding pattern is fabricated ormicrofabricated on the substrate” means the use of any microfabricationor micromachining methods to produce or generate encoding patterns onthe substrate. Various microfabrication or micromachining protocols suchas, pattern masking, photolithography, wet etching, reactive-ion-etchingand deep-reactive-ion-etching, etc., can be used.

As used herein, “major axis of the microdevice” refers to the longestdimension of the microdevice. For the microdevices having a thinround-disk shape, the height of the microdevice refers to the thicknessof the disk. In this case of thin round-disk shaped microdevices, themajor axis refers to any axis in the plane parallel to the majorsurfaces of the disk. In one preferred embodiment of such round-diskshaped microdevices, the photorecognizable coding patterns are on theplane parallel to the major surfaces of the disk surface, located on thedisk surface, or within the disk between the two major surfaces. For themicrodevices having a thin rectangular shape, three dimensions aredefined, the major axis (i.e., length), the minor axis (i.e., the width)and the height (i.e. the thickness of the rectangular microdevice). Insuch cases, the major axis of the microdevice is longer than the minoraxis and height of the microdevice. The minor axis of the microdevice islonger than or equals to the height of the microdevice. The microdevicesmay have any other shapes.

As used herein, “said microdevice has a preferential axis ofmagnetization” means that the induced magnetization of the microdeviceunder the influence of an applied magnetic field depends on the relativeangles of the direction of the applied magnetic field and various axesof the microdevices so that when the microdevices are introduced into aminimum-friction (or little- or no-friction) medium and/or placed on aminimum-friction (or little- or no-friction) surface, the microdevicemay rotate or orient itself under the interaction of the appliedmagnetic field and the induced magnetization to achieve a minimum energystate or stable state. When the microdevices introduced into a minimumfriction (or little or no-friction) medium and/or placed on aminimum-friction (or little- or no-friction) surface are in such aminimum energy state, the microdevice's axis that is aligned with theapplied magnetic field is the preferential axis of magnetization. Thepreferential axis of magnetization is determined by the geometry of themicrodevice, e.g., the ratio between the dimensions of the major axisand the minor axis, as well as the composition and structuralconfiguration of the microdevices. Depending on the geometry of themicrodevice, the preferential axis of magnetization can be a single axisin a particular direction or multiple axes in multiple directions, oreven any axis direction lying within a plane. Once the dynamic processof inducing magnetization is over and the microdevice has achieved theminimum energy state in a magnetic field, the induced magnetizationalong the preferential axis of magnetization (in its absolute magnitude)is larger than or at least equal to induced magnetization along anyother axis of the microdevice. In general, for the microdevices of thepresent invention to rotate or orient itself under the interaction ofthe applied magnetic field and the induced magnetization, the inducedmagnetization (in its absolute magnitude) along the preferential axis ofmagnetization of the microdevice should be at least 20% more than theinduced magnetization of the microdevice along at least one other axis.Preferably, the induced magnetization (in its absolute magnitude) alongthe preferential axis of magnetization of the microdevices of thepresent invention should be at least 50%, 70%, or 90% more than theinduced magnetization of the microdevice along at least one other axis.Even more preferably, the induced magnetization (in its absolutemagnitude) along the preferential axis of the magnetization of themicrodevices of the present invention should be at least one time,twice, five times, ten times, twenty times, fifty times or even hundredtimes more than the induced magnetization of the microdevice along atleast one other axis. The rotation and orientation of the microdeviceunder the influence of the applied magnetic field is a dynamic processand may take some time to achieve the minimum energy state or stablestate. In an environment where friction or other force, e.g., gravity,exists, the preferential axis of the magnetization of microdevice maynot align with the applied magnetic field perfectly even when asteady-state is achieved. Preferably, numerous factors such as thegeometry of the microdevice, the direction and strength of the appliedmagnetic field and other factors (e.g., for a microdevice lying on asupport surface, the frictional force that may relate to the property ofthe support surface may be a factor) can be adjusted to ensure that thepreferential axis of magnetization of microdevice is substantiallyaligned with the applied magnetic field when a steady-state is achieved.For example, for a microdevice having a thin round-disk shape withmagnetizable substance inside having a thin disc shape, the preferentialaxis of magnetization of such microdevice may lie in the plane parallelto the major surfaces of the microdevice (and also parallel to the majorsurface of the thin disk magnetic substance). When such a microdevice issubject to an applied magnetic field, even if initially the thin diskmicrodevice lies in the plane normal to the applied magnetic field, themicrodevice will re-align itself so that the thin disk plane will beparallel or close-to-parallel to the direction of the magnetic field. Inanother example, the microdevice has a thin rectangular shape insidewhich the magnetizable substance forms a magnetic structure such as amagnetic rectangular bar whose length, width and thickness are in thesame directions as those of the microdevice itself. The preferentialaxis of magnetization of such microdevice may be in the same directionas the length-direction of the microdevice and the length direction ofthe magnetic bar inside the microdevice.

As used herein, “the preferential axis of magnetization of themicrodevice is substantially aligned with an applied magnetic field”means that the angle between the preferential axis of the magnetizationand the applied magnetic field should be 45 degrees or less. Preferably,the angle between the preferential axis of magnetization and the appliedmagnetic field should be 15 degrees or less. More preferably, thepreferential axis of magnetization is completely aligned with theapplied magnetic field. For microdevices whose preferential axis ofmagnetization is the major axis, then “the preferential axis ofmagnetization of the microdevice is substantially aligned with anapplied magnetic field” means that the angle between the major axis ofthe microdevice and the applied magnetic field should be 45 degrees orless. For example, for microdevices having thin rectangular shape andhaving the major axis as the preferential axis of magnetization, anapplied magnetic field may result in the formation of a chain of themicrodevices along their major axises. When the applied magnetic fieldrotates for more than 45 degree (e.g. 90 degree), the microdevices wouldalso rotate for the same or similar degrees so that each microdevice inthe chain is substantially separated from each other.

As used herein, “the preferential axis of magnetization of themicrodevice is substantially aligned with microdevice's major axis”means that the angle between the preferential axis of the magnetizationand the major axis should be 45 degrees or less. Preferably, the anglebetween the preferential axis of magnetization and the major axis shouldbe 15 degrees or less. More preferably, the preferential axis ofmagnetization is completely aligned with the major axis.

As used herein, “each microdevice in the chain is substantiallyseparated from each other” means that the microdevices are sufficientlyseparated so that each of the microdevices can be identified and/oranalyzed by its respective photorecognizable coding pattern. The degreeof the separation among individual microdevices is determined by anumber of factors such as the type, number and/or distribution of thephotorecognizable coding pattern(s), the geometry of the microdevices,the methods for assessing the photorecognizable coding pattern(s) andthe purpose of the identification and/or analysis of the microdevices.Certain touch or overlap among individual microdevices are permissibleso long as each of the microdevices can be identified and/or analyzed byits respective photorecognizable coding pattern for the intendedpurpose. In certain situations, it is preferably that the microdevicesare completely separated from each other without any touching oroverlap.

As used herein, “said microchannels are sufficiently wide to permitrotation of said microdevices within said microchannels but sufficientlynarrow to prevent said microdevices from forming a chain when the majoraxis of said microdevices is substantially perpendicular to the majoraxis of said microchannels” means that the width of a microchannelequals to or is larger than the longest dimension of microdevices, e.g.,diagonal dimension of a rectangle, within the microchannel to permitrotation of the microdevices within the microchannel. At the same time,the width of a microchannel equals to or is less than 150% of thelongest dimension of microdevices, e.g., diagonal dimension of arectangle, within the microchannel to prevent microdevices from forminga chain (of at least two microdevices) when the major axis of saidmicrodevices is substantially perpendicular to the major axis of saidmicrochannels. Preferably, the width of a microchannel equals to or isless than 150%, 140%, 130%, 120%, 110%, or 105% 102% of the longestdimension of microdevices. “Sufficiently narrow to prevent saidmicrodevices from forming a chain” also means that after the rotation,each microdevice in the chain is substantially separated from each otheras defined above. It is not necessary, although permissible, that eachof the microchannels within a microchannel array has same width. It issufficient that each of the microchannels has a width that is compatibleto the microdevices to be rotated within the microchannel. Here, themajor axis of the microchannel refers to the length direction of themicrochannel.

As used herein, “the major axis of said microdevices is substantiallyperpendicular to the major axis of said microchannels” means that theangle between the major axis of microdevices and the major axis of themicrochannel that contains the microdevices equals to or is larger than45 degrees. Preferably, the angle between the major axis of microdevicesand the major axis of the microchannels that contains the microdevicesequals to or is larger than 50, 55, 60, 65, 70, 75, 80, 85 and 90degrees. Here, the major axis of the microchannels refers to the lengthdirection of the microchannels.

As used herein, “the height of the microchannels and/or the constrainton the microdevices by a magnetic field does not allow the microdevicesto stand up within the microchannels” means that the height of themicrochannels alone, the constraint on the microdevices by a magneticfield alone, or both, may be sufficient to prevent microdevices fromtaking a position so that the major axis of the microdevices issubstantially aligned with the height of the microchannel. In thesecases, the dimension of microchannel is defined by its length, width andheight. Its length corresponds to the major axis of the microchannel.The microchannel height corresponds to the microchannel axis that isnormal to the surface on which the microchannel is positioned. Themicrochannel width refers to the third dimension. The “major axis of themicrodevices is substantially aligned with the height of themicrochannel” means that the angle between the major axis of themicrodevices and the height of the microchannel equals to is less than45 degrees. When the constraint on the microdevices by a magnetic fieldalone is sufficient to prevent microdevices from taking such aprohibitive position, the height of the microchannels becomes irrelevantin this consideration.

As used herein, “said photorecognizable coding pattern corresponds to anentity to be synthesized on said microdevice” means that the entity tobe synthesized on a particular microdevice is predetermined according tothe photorecognizable coding pattern on that microdevice. The codingpattern can determine the entity to be synthesized on a microdevice indifferent ways. For example, a coding pattern can have multiple digitsand each digit determines a particular synthesis reaction and thecollection of all digits collectively determines all synthesisreactions, and hence the identity of the entity to be synthesized.Alternatively, a coding pattern can be an “intact” pattern, i.e., theentire pattern, not a portion or a digit of the pattern, determines theentire synthesis reactions on the microdevice, and hence the identity ofthe entity to be synthesized.

As used herein, “said microdevices are sorted after each synthesis cycleaccording to said photorecognizable coding patterns” means that thesynthetic steps or orders for making an entity on a particularmicrodevice are predetermined according to the photorecognizable codingpattern on that microdevice and after each synthesis cycle, thephotorecognizable coding pattern on the microdevice is assessed fordirecting the next synthetic step or order.

As used herein, “electrically conductive or dielectrically polarizablesubstance” refers to any substance that can be subjected todielectrophoresis force under appropriate conditions. Depending on thedielectric and electric properties of the substance, the substance maybe subject to positive or negative dielctrophpresis forces under certainconditions. Such conditions include, but are not limited to, thefrequency of the applied electric field, and the electrical anddielectric property of the medium in which the substance is placed orintroduced.

As used herein, “optical labeling substance” refers to any opticallydetectable substance that can be used to label the microdevices of thepresent invention to facilitate and/or enable detection and/oridentification of the microdevices. Quantum-dot is an example of anoptical labeling substance.

As used herein, “scattered-light detectable particle” refers to anyparticle that can emit unique and identifiable scattered-light uponillumination with light under appropriate conditions. The nano-sizedparticles with certain “resonance light scattering (RLS)” properties areexamples of one type of “scattered-light detectable particle”.

As used herein, “quantum dot” refers to a fluorescent label comprisingwater-soluble semiconductor nanocrystal(s). One unique feature of aquantum dot is that its fluorescent spectrum is related or determined bythe diameter of its nanocrystals(s). “Water-soluble” is used herein tomean sufficiently soluble or suspendable in a aqueous-based solution,such as in water or water-based solutions or physiological solutions,including those used in the various fluorescence detection systems asknown by those skilled in the art. Generally, quantum dots can beprepared which result in relative monodispersity; e.g., the diameter ofthe core varying approximately less than 10% between quantum dots in thepreparation.

As used herein, “chip” refers to a solid substrate with a plurality ofone-, two- or three-dimensional micro structures or micro-scalestructures on which certain processes, such as physical, chemical,biological, biophysical or biochemical processes, etc., can be carriedout. The micro structures or micro-scale structures such as, channelsand wells, electrode elements, electromagnetic elements, areincorporated into, fabricated on or otherwise attached to the substratefor facilitating physical, biophysical, biological, biochemical,chemical reactions or processes on the chip. The chip may be thin in onedimension and may have various shapes in other dimensions, for example,a rectangle, a circle, an ellipse, or other irregular shapes. The sizeof the major surface of chips used in the present invention can varyconsiderably, e.g., from about 1 mm² to about 0.25 m². Preferably, thesize of the chips is from about 4 mm² to about 25 cm² with acharacteristic dimension from about 1 mm to about 7.5 cm. The chipsurfaces may be flat, or not flat. The chips with non-flat surfaces mayinclude channels or wells fabricated on the surfaces.

As used herein, “a means for generating a physical force on said chip”refers to any substance, structure or a combination thereof that iscapable of generating, in conjunction with an built-in structure on achip, to generate a desirable physical force on the chip.

As used herein, “physical field,” e.g., used itself or used as “physicalfield in a region of space” or “physical field is generated in a regionof space” means that the region of space has following characteristics.When a moiety, alone or bound to a microdevice, of appropriateproperties is introduced into the region of space (i.e. into thephysical field), forces are produced on the moiety, the microdevice orboth, as a result of the interaction between the moiety and/ormicrodevice and the field. A moiety can be manipulated within a fieldvia the physical forces exerted on the moiety by the field. Exemplaryfields include electric, magnetic, acoustic, optical and velocityfields. In the present invention, physical field always exists in amedium in a region of space, and the moiety to be manipulated issuspended in, or is dissolved in, or more generally, is placed in themedium. Typically, the medium is a fluid such as aqueous or non-aqueousliquids, or a gas. Depending on the field configuration, an electricfield may produce electrophoretic forces on charged moieties, or mayproduce conventional dielectrophoretic forces and/or traveling wavedielectrophoretic forces on charged and/or neutral moieties. Magneticfield may produce magnetic forces on magnetic moieties. Acoustic fieldmay produce acoustic radiation forces on moieties. Optical field mayproduce optical radiation forces on moieties. Velocity field in themedium in a region of space refers to a velocity distribution of themedium that moves in the region of the space. Various mechanisms may beresponsible for causing the medium to move and the medium at differentpositions may exhibit different velocities, thus generating a velocityfield. Velocity field may exert mechanical forces on moieties in themedium.

As used herein, “medium (or media)” refers to a fluidic carrier, e.g.,liquid or gas, wherein a moiety, alone or bound to a microdevice, isdissolved, suspended or contained.

As used herein, “microfluidic application” refers to the use ofmicroscale devices, e.g., the characteristic dimension of basicstructural elements is in the range between less than 1 micron to 1 cmscale, for manipulation and process in a fluid-based setting, typicallyfor performing specific biological, biochemical or chemical reactionsand procedures. The specific areas include, but are not limited to,biochips, i.e., chips for biologically related reactions and processes,chemchips, i.e., chips for chemical reactions, or a combination thereof.The characteristic dimensions of the basic elements refer to the singledimension sizes. For example, for the microscale devices having circularshape structures (e.g. round electrode pads), the characteristicdimension refers to the diameter of the round electrodes. For thedevices having thin, rectangular lines as basic structures, thecharacteristic dimensions may refer to the width or length of theselines.

As used herein, “built-in structures on said substrate of a chip” meansthat the structures are built into the substrate or the structures arelocated on the substrate or the structures are structurally linked tothe substrate of the chip. In one embodiment, the built-in structuresmay be fabricated on the substrate. For example, as described in“Dielectrophoretic manipulation of cells using spiral electrodes by Wanget al., Biophys. J., 72:1887-1899 (1997)”, spiral electrodes arefabricated on a glass substrate. Here the spiral electrodes are“built-in” structures on the glass substrate. In another embodiment, the“built-in” structures may be first fabricated on one substrate and thestructure-containing first substrate may then be attached or bound to asecond substrate. Such structures are “built-in” structures not only onthe first substrate but also on the second substrate. In still anotherembodiment, the built-in structures may be attached or bound to thesubstrate. For example, thin, electrically-conductive wires may be usedas electrodes for producing electric field. These electric wires may bebound or attached to a glass substrate. In this case, theelectrically-conductive wires are “built-in” structures on the glasssubstrate. Throughout this application, when it is described that“built-in” structures on the chip or on the substrate are capable ofgenerating physical forces and/or physical fields or these structuresgenerate physical forces and/or physical fields, these structures areused in combination with external signal sources or external energysources.

As used herein, “micro-scale structures” means that the scale of theinternal structures of the apparatus for exerting desired physicalforces must be compatible with and useable in microfluidic applicationsand have characteristic dimension of basic structural elements in therange from about 1 micron to about 20 mm scale.

As used herein, “moiety” refers to any substance whose isolation,manipulation, measurement, quantification, detection or synthesis usingthe present microdevice is desirable. Normally, the dimension (or thecharacteristic dimensions) of the moiety should not exceed 1 cm. Forexample, if the moiety is spherical or approximately spherical, thedimension of the moiety refers to the diameter of the sphere or anapproximated sphere for the moiety. If the moiety is cubical orapproximately cubical, then the dimension of the moiety refers to theside width of the cube or an approximated cube for the moiety. If themoiety has an irregular shape, the dimension of the moiety may refer tothe average between its largest axis and smallest axis. Non-limitingexamples of moieties include cells, cellular organelles, viruses,particles, molecules, e.g., proteins, DNAs and RNAs, or an aggregate orcomplex thereof.

Moiety to be isolated, manipulated, measured, quantified, detected orsynthesized includes many types of particles—solid (e.g., glass beads,latex particles, magnetic beads), liquid (e.g., liquid droplets) orgaseous particles (e.g., gas bubble), dissolved particles (e.g.,molecules, proteins, antibodies, antigens, lipids, DNAs, RNAs,molecule-complexes), suspended particles (e.g., glass beads, latexparticles, polystyrene beads). Particles can be organic (e.g., mammaliancells or other cells, bacteria, virus, or other microorganisms) orinorganic (e.g., metal particles). Particles can be of different shapes(e.g., sphere, elliptical sphere, cubic, discoid, needle-type) and canbe of different sizes (e.g., nano-meter-size gold sphere, tomicrometer-size cells, to millimeter-size particle-aggregate). Examplesof particles include, but are not limited to, biomolecules such as DNA,RNA, chromosomes, protein molecules (e.g., antibodies), cells, colloidparticles (e.g., polystyrene beads, magnetic beads), any biomolecules(e.g., enzyme, antigen, hormone etc).

As used herein, “plant” refers to any of various photosynthetic,eucaryotic multi-cellular organisms of the kingdom Plantae,characteristically producing embryos, containing chloroplasts, havingcellulose cell walls and lacking locomotion.

As used herein, “animal” refers to a multi-cellular organism of thekingdom of Animalia, characterized by a capacity for locomotion,nonphotosynthetic metabolism, pronounced response to stimuli, restrictedgrowth and fixed bodily structure. Non-limiting examples of animalsinclude birds such as chickens, vertebrates such fish and mammals suchas mice, rats, rabbits, cats, dogs, pigs, cows, ox, sheep, goats,horses, monkeys and other non-human primates.

As used herein, “bacteria” refers to small prokaryotic organisms (lineardimensions of around 1 micron) with non-compartmentalized circular DNAand ribosomes of about 70S. Bacteria protein synthesis differs from thatof eukaryotes. Many anti-bacterial antibiotics interfere with bacteriaproteins synthesis but do not affect the infected host.

As used herein, “eubacteria” refers to a major subdivision of thebacteria except the archaebacteria. Most Gram-positive bacteria,cyanobacteria, mycoplasmas, enterobacteria, pseudomonas and chloroplastsare eubacteria. The cytoplasmic membrane of eubacteria containsester-linked lipids; there is peptidoglycan in the cell wall (ifpresent); and no introns have been discovered in eubacteria.

As used herein, “archaebacteria” refers to a major subdivision of thebacteria except the eubacteria. There are three main orders ofarchaebacteria: extreme halophiles, methanogens and sulphur-dependentextreme thermophiles. Archaebacteria differs from eubacteria inribosomal structure, the possession (in some case) of introns, and otherfeatures including membrane composition.

As used herein, “virus” refers to an obligate intracellular parasite ofliving but non-cellular nature, consisting of DNA or RNA and a proteincoat. Viruses range in diameter from about 20 to about 300 nm. Class Iviruses (Baltimore classification) have a double-stranded DNA as theirgenome; Class II viruses have a single-stranded DNA as their genome;Class III viruses have a double-stranded RNA as their genome; Class IVviruses have a positive single-stranded RNA as their genome, the genomeitself acting as mRNA; Class V viruses have a negative single-strandedRNA as their genome used as a template for mRNA synthesis; and Class VIviruses have a positive single-stranded RNA genome but with a DNAintermediate not only in replication but also in mRNA synthesis. Themajority of viruses are recognized by the diseases they cause in plants,animals and prokaryotes. Viruses of prokaryotes are known asbacteriophages.

As used herein, “fungus” refers to a division of eucaryotic organismsthat grow in irregular masses, without roots, stems, or leaves, and aredevoid of chlorophyll or other pigments capable of photosynthesis. Eachorganism (thallus) is unicellular to filamentous, and possesses branchedsomatic structures (hyphae) surrounded by cell walls containing glucanor chitin or both, and containing true nuclei.

As used herein, “binding partners” refers to any substances that bind tothe moieties with desired affinity or specificity. Non-limiting examplesof the binding partners include cells, cellular organelles, viruses,particles, microparticles or an aggregate or complex thereof, or anaggregate or complex of molecules, or specific molecules such asantibodies, single stranded DNAs. The binding partner can be a substancethat is coated on the surface of the present microdevice. Alternatively,the binding partner can be a substance that is incorporated, e.g.,microfabricated, into the material composition of the presentmicrodevice. The material composition of the present microdevice, inaddition being a substrate, may possess binding affinity to certainmoiety, and thus functioning as a binding partner itself.

As used herein, “an element that facilitates and/or enables manipulationof the microdevice and/or a moiety/microdevice complex” refers to anysubstance that is itself manipulatable or makes the moiety/microdevicecomplex manipulatable with the desired physical force(s). Non-limitingexamples of the elements include cells, cellular organelles, viruses,particles, microparticles or an aggregate or complex thereof, or anaggregate or complex of molecules. Non-limiting examples of the elementsmay further include deposited or other-procedure-produced materials withspecific physical or chemical properties. Metal films made of Au, Cr,Ti, Pt etc are examples of the elements that can be incorporated intothe microdevices and increase electrical conductivity of themicrodevices. Insulating materials such as polystyrene, paralene, orother plastic polymers are also examples of the elements that may beincorporated into the microdevices and reduce electrical conductivity ofthe microdevices.

As used herein, “microparticles” refers to particles of any shape, anycomposition, any complex structures that are manipulatable by desiredphysical force(s) in microfluidic settings or applications. One exampleof microparticles is magnetic beads that are manipulatable by magneticforces. Another example of a microparticle is a cell that ismanipulatable by an electric force such as a traveling-wavedielectrophoretic force. The microparticles used in the methods can havea dimension from about 0.01 micron to about ten centimeters. Preferably,the microparticles used in the methods have a dimension from about 0.01micron to about several thousand microns. Examples of the microparticlesinclude, but are not limited to, plastic particles, polystyrenemicrobeads, glass beads, magnetic beads, hollow glass spheres, particlesof complex compositions, microfabricated free-standing microstructures,etc. Other particles include cells, cell organelles, large biomoleculessuch as DNA, RNA and proteins etc.

As used herein, “manipulation” refers to moving or processing of themoieties, and the microdevices disclosed in the present invention, whichresults in one-, two- or three-dimensional movement of the moiety (andthe microdevices). The manipulation can be conducted off a chip or in achip format, whether within a single chip or between or among multiplechips, or on a substrate or among substrates of an apparatus.“Manipulation” of moieties and the microdevices can also be performed inliquid containers. Non-limiting examples of the manipulations includetransportation, focusing, enrichment, concentration, aggregation,trapping, repulsion, levitation, separation, sorting, fractionation,isolation or linear or other directed motion of the moieties. Foreffective manipulation, the characteristics of the moiety (and themicrodevices) to be manipulated and the physical force used formanipulation must be compatible. For example, the microdevices withcertain magnetic properties can be used with magnetic force. Similarly,the microdevices with electric charge(s) can be used with electrostatic(i.e. electrophoretic) force. In the case of manipulatingmicrodevices-binding partner-moiety complexes, the characteristics ofthe moiety, or its binding partner or the microdevices, and the physicalforce used for manipulation must be compatible. For example, moiety orits binding partner or the microdevices with certain dielectricproperties to induce dielectric polarization in the moiety or itsbinding partner or the microdevices can be used with dielectrophoresisforce.

As used herein, “the moiety is not directly manipulatable” by aparticular physical force means that no observable movement of themoiety can be detected when the moiety itself not coupled to a bindingpartner is acted upon by the particular physical force.

As used herein, “physical force” refers to any force that moves themoieties or their binding partners or the corresponding microdeviceswithout chemically or biologically reacting with the moieties and thebinding partners, or with minimal chemical or biological reactions withthe binding partners and the moieties so that the biological/chemicalfunctions/properties of the binding partners and the moieties are notsubstantially altered as a result of such reactions. Throughout theapplication, the term of “forces” or “physical forces” always means the“forces” or “physical forces” exerted on a moiety or moieties, thebinding partner(s) and/or the microdevice(s). The “forces” or “physicalforces” are always generated through “fields” or “physical fields”. Theforces exerted on moieties, the binding partner(s) and/or themicrodevice(s) by the fields depend on the properties of the moieties,the binding partner(s) and/or the microdevice(s). Thus, for a givenfield or physical field to exert physical forces on a moiety, it isnecessary for the moiety to have certain properties. While certain typesof fields may be able to exert forces on different types of moietieshaving different properties, other types of fields may be able to exertforces on only limited type of moieties. For example, magnetic field canexert forces or magnetic forces only on magnetic particles or moietieshaving certain magnetic properties, but not on other particles, e.g.,polystyrene microdevices. On the other hand, a non-uniform electricfield can exert physical forces on many types of moieties such aspolystyrene microdevices, cells, and also magnetic particles. It is notnecessary for the physical field to be able to exert forces on differenttypes of moieties or different moieties. But it is necessary for thephysical field to be able to exert forces on at least one type of moietyor at least one moiety, the binding partner(s) and/or themicrodevice(s).

As used here in, “electric forces (or electrical forces)” are the forcesexerted on moieties, the binding partner(s) and/or the microdevice(s) byan electric (or electrical) field.

As used herein, “magnetic forces” are the forces exerted on moieties,the binding partner(s) and/or the microdevice(s) by a magnetic field.

As used herein, “acoustic forces (or acoustic radiation forces)” are theforces exerted on moieties, the binding partner(s) and/or themicrodevice(s) by an acoustic field.

As used herein, “optical (or optical radiation) forces” are the forcesexerted on moieties, the binding partner(s) and/or the microdevice(s) byan optical field.

As used herein, “mechanical forces” are the forces exerted on moieties,the binding partner(s) and/or the microdevice(s) by a velocity field.

As used herein, “sample” refers to anything which may contain a moietyto be isolated, manipulated, measured, quantified or detected by thepresent microdevices and/or methods. The sample may be a biologicalsample, such as a biological fluid or a biological tissue. Examples ofbiological fluids include urine, blood, plasma, serum, saliva, semen,stool, sputum, cerebral spinal fluid, tears, mucus, amniotic fluid orthe like. Biological tissues are aggregates of cells, usually of aparticular kind together with their intercellular substance that formone of the structural materials of a human, animal, plant, bacterial,fungal or viral structure, including connective, epithelium, muscle andnerve tissues. Examples of biological tissues also include organs,tumors, lymph nodes, arteries and individual cell(s). The sample mayalso be a mixture of target analyte or enzyme containing moleculesprepared in vitro.

As used herein, a “liquid (fluid) sample” refers to a sample thatnaturally exists as a liquid or fluid, e.g., a biological fluid. A“liquid sample” also refers to a sample that naturally exists in anon-liquid status, e.g., solid or gas, but is prepared as a liquid,fluid, solution or suspension containing the solid or gas samplematerial. For example, a liquid sample can encompass a liquid, fluid,solution or suspension containing a biological tissue.

As used herein the term “assessing (or assessed)” is intended to includequantitative and qualitative determination of the identity and/orquantity of a moiety, e.g., a protein or nucleic acid, present in thesample or on the microdevices or in whatever form or state. Assessmentwould involve obtaining an index, ratio, percentage, visual or othervalue indicative of the identity of a moiety in the sample and mayfurther involve obtaining a number, an index, or other value indicativeof the amount or quantity or the concentration of a moiety present inthe sample or on the microdevice or in whatever form or state.Assessment may be direct or indirect.

B. Microdevices and Systems for Forming a Microdevice Array

In one aspect, the present invention is directed to a microdevice, whichmicrodevice comprises: a) a magnetizable substance; and b) aphotorecognizable coding pattern, wherein said microdevice has apreferential axis of magnetization.

Any suitable magnetizable substance can be used in the presentmicrodevices. In one example, the magnetizable substance used in themicrodevice is a paramagnetic substance, a ferromagnetic substance, aferrimagnetic substance, or a superparamagnetic substance. In anotherexample, the magnetizable substance used in the microdevice comprises ametal composition. Preferably, the metal composition is a transitionmetal composition or an alloy thereof such as iron, nickel, copper,cobalt, manganese, tantalum, zirconium and cobalt-tantalum-zirconium(CoTaZr) alloy. In a preferred example, the magnetic substance is ametal oxide Fe₃O₄.

The present microdevice can further comprise a non-magnetizablesubstrate. Any suitable material including silicon, plastic, glass,ceramic, rubber, polymer, silicon dioxide, silicon nitride, aluminumoxide, titanium, aluminum, gold and a combination thereof can be used inthe substrate. The magnetizable substance can be linked to the substratein any form. For example, the magnetizable substance can be made part ofthe substrate or can be attached or deposited or located on thesubstrate. In another example, the magnetizable substance can be locatedwithin the substrate.

The substrate can be a single layer or can comprise multiple layers suchas 3, 4 or more layers. For example, a substrate can have 3 layers. Thetop and the bottom layers can be made of the same material, e.g., SiO₂(or glass) and the middle layer can contain magnetizable material(s).Alternatively, the top and the bottom layers can have differentmaterials.

The substrate can comprises a surface that is hydrophobic orhydrophilic. The substrate can be in any suitable shape such asrectangle and other regular or irregular shape provided that themicrodevice be made to have a preferential axis of magnetization. Thesubstrate can be in any suitable dimension(s). For example, thethickness of the substrate can be from about 0.1 micron to about 500microns. Preferably, the thickness of the substrate can be from about 1micron to about 200 microns. More preferably, the thickness of thesubstrate can be from about 1 micron to about 50 microns. In a specificembodiment, the substrate is a rectangle having a surface area fromabout 10 squared-microns to about 1,000,000 squared-microns (e.g., 1000micron by 1000 micron). In another specific embodiment, the substrate isin an irregular shape having a single-dimension from about 1 micron toabout 500 microns. In a preferred embodiment, the substrate is acomposite comprising silicon, metal film and polymer film. In anotherpreferred embodiment, the substrate can comprise a silicon layer and ametal layer, e.g., an aluminum layer. More preferably, the metal layercan comprise a magnetic material, such as nickel metal or CoTaZr(Cobalt-Tantalum-Zirconium) alloy.

The photorecognizable coding pattern can be based on any suitablephotorecognizable (optical) property constructed in or on themicrodevice or substrate. For example, the photorecognizable codingpattern can be the material composition of the microdevice itself, ahole in the microdevice, or other structural configurations, or certainsubstance(s) located, deposited or immobilized on the microdevice or thesubstrate, or an optical labeling substance or an 1-D and/or a 2-D barcoding pattern. The microdevice or substrate can be patterned. Inaddition, the surface layer of the substrate or microdevice can bemodified. The versatility of the photorecognizable coding pattern can becaused by the shape, number, letters, words, position distribution,optical refractive property, material composition, or a combinationthereof, of the substrate, the hole(s) or other structureconfigurations, or certain substance(s) located, deposited orimmobilized on the microdevice or the substrate. In one exemplarymicrodevice, the microdevice or substrate can have 4 layers. The top andthe bottom layers can be made of the same material, e.g., SiO₂ (orglass). One of the middle layers can contain paramagnetic material(s),e.g., magnetic alloys. The other middle layer can contain aphotorecognizable coding pattern as a encoding layer. Preferably, theparamagnetic layer and the encoding layer do not substantially overlap,or do not overlap at all, to ensure optical detection of thephotorecognizable coding pattern in the encoding layer. Alternatively,the top and the bottom layers can have different materials. Exemplarypatterns include numbers, letters, structures, 1-D and 2-D barcodes.

Although the microdevice can comprise a single photorecognizable codingpattern, it can also comprise a plurality of photorecognizable codingpatterns, e.g., a plurality of holes or other structure configurations,a plurality of numbers, a plurality of letter, and/or a plurality of thesubstances.

To facilitate optical analysis (or photo-analysis) of encoding patterns,certain microdevices may incorporate “orientation” marks or alignmentmarkers. For example, for the microdevices having thin symmetricalshapes, the microdevices lying flat on either of its major surfaces willlook identical, causing difficulties in identification. Therefore, theorientation marks can be used for indicating which major surface isbeing looked at when the microdevices are lying up and for helpingdecode the patterns.

The photorecognizable coding pattern can be constructed according to anymethods known in the art. For example, the photorecognizable codingpattern can be fabricated or microfabricated on a substrate. Anysuitable fabrication or microfabrication method can be used includinglithography such as photolithography, electron beam lithography andX-ray lithography (WO 96/39937 and U.S. Pat. Nos. 5,651,900, 5,893,974and 5,660,680). For example, the fabrication or microfabrication methodscan be used directly on a substrate to produce desirable patterns suchas numbers, letters, structures, 1-D and 2-D barcodes.

If a substance having an optical refractive property that is differentfrom the optical refractive property of the substrate is used as thephotorecognizable coding pattern, the substance can be deposited orimmobilized on the substrate by any suitable methods known in the art.For example, the substance used for photorecognizable encoding can bedeposited or immobilized on the substrate by evaporation or sputteringmethods. The substance can be deposited or immobilized on the substratedirectly or via a linker. The linker can be any material or moleculesthat linking the substance to the substrate. The fabrication ormicrofabrication methods can be used on the substances deposited on thesubstrate to produce desirable patterns such as numbers, letters,structures, 1-D and 2-D barcodes. The substance can be immobilized ordeposited on the substrate via a covalent or a non-covalent linkage. Thesubstance can be deposited or immobilized on the substrate via specificor non-specific binding.

Any suitable optical labeling substance can be used in the presentmicrodevices. In a specific embodiment, the optical labeling substanceused in the present microdevices is a metal film such as Cu, Al, Au, Ptthat can be patterned to form photorecognizable encoding patters such asletters, numbers, structures or structural configurations, 1-D or 2-Dbarcodes. In another specific embodiment, the optical labeling substanceused in the present microdevices is a fluorescent substance, ascattered-light detectable particle (See e.g., U.S. Pat. No. 6,214,560)and a quantum dot (See e.g., U.S. Pat. No. 6,252,664).

Any suitable quantum dot can be used in the present microdevices. In aspecific embodiment, the quantum dot used in the present microdevicescomprises a Cd—X core, X being Se, S or Te. Preferably, the quantum dotcan be passivated with an inorganic coating shell, e.g., a coating shellcomprising Y-Z, Y being Cd or Zn, and Z being S or Se. Also preferably,the quantum dot can comprise a Cd—X core, X being Se, S or Te, a Y-Zshell, Y being Cd or Zn, and Z being S or Se, and the quantum dot canfurther be overcoated with a trialkylphosphine oxide.

Any suitable methods can be used to make the CdX core/YZ shell quantumdots water-soluble (See e.g., U.S. Pat. No. 6,252,664). One method tomake the CdX core/YZ shell quantum dots water-soluble is to exchange theovercoating layer with a coating which will make the quantum dotswater-soluble. For example, a mercaptocarboxylic acid may be used toexchange with the trialkylphosphine oxide coat. Exchange of the coatinggroup is accomplished by treating the water-insoluble quantum dots witha large excess of neat mercaptocarboxylic acid. Alternatively, exchangeof the coating group is accomplished by treating the water-insolublequantum dots with a large excess of mercaptocarboxylic acid in CHCl₃solution (Chan and Nie, 1998, Science 281:2016-2018). The thiol group ofthe new coating molecule forms Cd (or Zn)—S bonds, creating a coatingwhich is not easily displaced in solution. Another method to make theCdX core/YZ shell quantum dots water-soluble is by the formation of acoating of silica around the dots (Bruchez, Jr. et al., 1998, Science281:2013-2015). An extensively polymerized polysilane shell impartswater solubility to nanocrystalline materials, as well as allowingfurther chemical modifications of the silica surface. Generally, these“water-soluble” quantum dots require further functionalization to makethem sufficiently stable in an aqueous solution for practical use in afluorescence detection system (See e.g., U.S. Pat. No. 6,114,038),particularly when exposed to air (oxygen) and/or light. Water-solublefunctionalized nanocrystals are extremely sensitive in terms ofdetection, because of their fluorescent properties (e.g., including, butnot limited to, high quantum efficiency, resistance to photobleaching,and stability in complex aqueous environments); and comprise a class ofsemiconductor nanocrystals that may be excited with a single peakwavelength of light resulting in detectable fluorescence emissions ofhigh quantum yield and with discrete fluorescence peaks (e.g., having anarrow spectral band ranging between about 10 nm to about 60 nm).

The quantum dot used in the present microdevice can have any suitablesize. For example, the quantum dot can have a size ranging from about 1nm to about 100 nm.

The microdevice of the present invention can comprise a single quantumdot. Alternatively, the microdevice of the present invention cancomprise a plurality of quantum dots. Preferably, the microdevice of thepresent invention comprises at least two quantum dots that havedifferent sizes.

The microdevice of the present invention can comprise a single opticallabeling substance. Alternatively, the microdevice of the presentinvention can comprise a plurality of optical labeling substances. Forexample, the microdevice of the present invention can comprise at leasttwo different types of optical labeling substances.

In a specific embodiment, the microdevice of the present inventioncomprises an electrically conductive or dielectrically polarizablesubstance. Such electrically conductive or dielectrically polarizablesubstance incorporated into the microdevice may alter the overallelectrical and/or dielectric properties of the microdevice, resulting ina change in the interaction between the microdevice and an appliedelectrical field and a change in the electrical field-induced force(e.g., dielectrophoretic force, traveling wave dielectrophoretic forces)acting on the microdevice.

In choosing the type, materials, compositions, structures and sizes ofthe microdevices, these properties or parameters of the microdevicesshould be compatible with the isolation, manipulation, detection orsynthesis format in the specific applications. For example, themicrodevices may be used to isolate target analyte-molecules (e.g.proteins) from a molecule mixture. If the isolation usesdielectrophoretic forces, then the microdevices should have the desireddielectric properties. If the isolation/manipulation utilizes magneticforces, then the microdevices should have incorporated magneticmaterials such as ferro- or ferri-magnetic materials.

The microdevice can also comprise a binding partner that is capable ofbinding to a moiety, e.g., a moiety to be isolated, manipulated,detected or synthesized. Preferably, the binding partner specificallybinds to the moiety. Throughout this application, whenever the bindingpartners are described or used, they are always coupled onto themicrodevices of the present inventions. For example, when the complexesbetween the binding partners and the moieties are discussed, thecomplexes between the moieties and the binding partners that are coupledon the microdevices are referred to.

Any suitable binding partner including the binding partners disclosed inthe co-pending U.S. patent application Ser. No. 09/636,104, filed Aug.10, 2000 and 09/679,024, filed Oct. 4, 2000, the disclosures of whichare incorporated by reference in its entirety, can be used. For example,the binding partners can be cells such as animal, plant, fungus orbacterium cells; cellular organelles such as nucleus, mitochondria,chloroplasts, ribosomes, ERs, Golgi apparatuses, lysosomes, proteasomes,secretory vesicles, vacuoles or microsomes; viruses, microparticles oran aggregate or complex thereof. Other binding partners may be moleculesthat have been immobilized on the microdevices' surfaces. For example,antibodies can be immobilized or bound on to the microdevices' surfaces.The antibody-bound microdevices can then be used to capture and bind totarget proteins in a molecule mixture or to capture and bind to targetcells in a cell mixture. Oligo-dT (e.g. 25 mer of T) can be immobilizedonto the microdevices' surfaces. The oligo-dT bound microdevices canthen be used to capture mRNA from a molecule mixture. Other moleculesmay be used as binding partners for capturing or binding DNA molecules.Nucleic acid fragments, e.g., DNA, RNA, PNA segments of specificsequences, may be used to hybridize to target nucleic acid, DNA, RNA orPNA, molecule. Other binding partners may be molecules or functionalgroups that are attached or otherwise bound to the microdevices'surfaces, resulting in functionalized surfaces to which variouschemical/biochemical/biological reactions can occur. In someembodiments, these various reactions may allow the moieties to bind tothe microdevices so that the moieties can be manipulated, isolated, ordetected via the use of the microdevices of the present invention. Insome other exemplary embodiments, the functionalized surfaces allowsynthesis reaction to take place on the microdevices' surfaces. Examplesof such synthesis include the synthesis of nucleic acids, (e.g. DNA,RNA), or the synthesis of peptides or proteins, etc. Examples of suchfunctionalized surfaces include, but are not limited to, surfacesderivatized with carboxyl, amino, hydroxyl, sulfhydryl, epoxy, ester,alkene, alkyne, alkyl, aromatic, aldehyde, ketone, sulfate, amide,urethane group(s), or their derivatives thereof.

The choice of the microdevices is further related to the specificisolation, manipulation detection or synthesis uses. For example, forseparating target moiety from a mixture of molecules and particles bydielectrophoresis manipulation, binding partner's or microdevice'sdielectric properties should be significantly different from those ofmolecules and particles so that when binding partners are coupled withthe target moiety, the moiety-binding-partner-microdevices complexes maybe selectively manipulated by dielectrophoresis. In an example ofseparating target cancer cells from a mixture of normal cells, thecancer cells may have similar dielectric properties to those of normalcells and all the cells behave similarly in their dielectrophoreticresponses, e.g., negative dielectrophoresis. In this case, the bindingpartners or the microdevice preferably should be moredielectrically-polarizable than their suspending medium and will exhibitpositive dielectrophoresis. Thus, such microdevices-bindingpartners-cancer-cell complexes can be selectively manipulated throughpositive dielectrophoresis forces while other cells experience negativedielectrophoresis forces.

The microdevice can comprise a single binding partner. Alternatively, itcan be used in a high throughput analysis and can comprise a pluralityof binding partners capable of binding or specifically binding todifferent moieties to be isolated, manipulated or detected, orsynthesized.

Since the present microdevice contains magnetizable substance, themicrodevice, microdevice-moiety complex, or microdevice-bindingpartner-moiety complex can always be rotated or otherwise moved ormanipulated with magnetic forces. Magnetic forces refer to the forcesacting on a particle due to the application of a magnetic field. Ingeneral, particles have to be magnetic (e.g. paramagnetic,ferromagnetic) or magnetizable when sufficient magnetic forces areneeded to manipulate particles. We consider a typical magnetic particlemade of super-paramagnetic material. When the magnetic particle issubjected to a magnetic field B, a magnetic dipole μ is induced in themagnetic particle

$\begin{matrix}{{\overset{\_}{\mu} = {{V_{p}\left( {\chi_{p} - \chi_{m}} \right)}\frac{\overset{\_}{B}}{\mu_{m}}}},} \\{= {{V_{p}\left( {\chi_{p} - \chi_{m}} \right)}{\overset{\_}{H}}_{m}}}\end{matrix}$where V_(p) is the magnetic-particle volume, χ_(p) and χ_(m) are thevolume susceptibility of the magnetic particle and its surroundingmedium, μ_(m) is the magnetic permeability of medium, H _(m) is themagnetic field strength. The magnetic force F _(magnetic) acting on themagnetic particle is determined by the magnetic dipole moment and themagnetic field gradient:F _(magnetic)=−0.5V _(p)(χ_(p)−χ_(m)){right arrow over (H)} _(m)•∇{right arrow over (B)} _(m),where the symbols “•” and “∇” refer to dot-product and gradientoperations, respectively. Clearly, whether there is magnetic forceacting on a particle depends on the difference in the volumesusceptibility between the magnetic particle and its surrounding medium.Typically, magnetic particles are suspended in a liquid, non-magneticmedium (the volume susceptibility is close to zero) thus it is necessaryto utilize magnetic particles (its volume susceptibility is much largerthan zero). The velocity ν_(particle) of the magnetic particle under thebalance between magnetic force and viscous drag is given by:

$v_{particle} = \frac{{\overset{\_}{F}}_{magnetic}}{6\pi\; r\;\eta_{m}}$where r is the particle radius and η_(m) is the viscosity of thesurrounding medium. Thus to achieve sufficiently large magneticmanipulation force, the following factors should be considered: (1) thevolume susceptibility of the magnetic particles should be maximized; (2)magnetic field strength should be maximized; and (3) magnetic fieldstrength gradient should be maximized.

Paramagnetic substances are preferred whose magnetic dipoles are inducedby externally applied magnetic fields and return to zero when externalfield is turned off. Examples of the paramagnetic substances include thecommercially available paramagnetic or other magnetic particles. Many ofthese particles range from submicron (e.g., 50 nm-0.5 micron) up to tensof microns. They may have different structures and compositions. Onetype of magnetic particle has ferromagnetic materials encapsulated inthin polymer layer, e.g., polystyrene. Another type of magnetic particlehas ferromagnetic nanoparticles filled into the poles of porous beadse.g., polystyrene beads. The surface of both types of these particlescan be polystyrene in nature and may be modified to link to varioustypes of molecules. In still another type of magnetic particle,ferro-magnetic materials can be incorporated uniformly into theparticles during the polymerization process. Thus, in certainembodiments of the microdevices of the present invention, theseparamagnetic or magnetic particles may be incorporated into themicrodevices so that the microdevices comprise the magnetizablesubstances.

Exemplary embodiments of the magnetizable substance comprised in themicrodevices may include paramagnetic substance, ferromagneticsubstance, ferrimagnetic substance, or superparamagnetic substance thatare directly deposited or fabricated or incorporated into themicrodevices. In one example, the metal composition such as transitionmetal composition (e.g., iron, nickel, copper, cobalt, manganese,tantalum, zirconium) or an alloy (e.g., cobalt-tantalum-zirconium(CoTaZr) alloy, iron-nickel alloy) composition may be deposited into themicrodevices. Various methods such as electroplating (e.g., for makingiron-nickel alloy), sputtering (e.g. for making CoTaZr alloy), can beused for depositing magnetizable substances. A number of methods fordepositing and/or producing magnetizable substances (e.g. magnetic,paramagnetic, ferro magnetic substances) are described in the U.S.patent application Ser. No. 09/685,410, filed on Oct. 10, 2000, titled“Individually Addressable Micro-Electromagnetic Unit Array Chips inHorizontal Configurations” and naming Wu et al as inventors. This patentapplication Ser. No. 09/685,410 is incorporated by reference in itsentirety.

The rotation or manipulation of the microdevices, microdevice-moietycomplex, or microdevice-binding partner-moiety complex, requires thegeneration of magnetic field distribution over microscopic scales. Onedesirable feature of a microdevice is that it has large magneticsusceptibility. Another desirable feature is that it has small residuemagnetic polarization after the applied magnetic field/force is turnedoff. One approach for generating such magnetic fields is the use ofmicroelectromagnetic units. Such units can induce or produce magneticfields when an electrical current is applied. The on/off status and themagnitude of the electrical current applied to each unit will determinethe magnetic field distribution. The structure and dimension of themicroelectromagnetic units may be designed according to the requirementof the magnetic field distribution. Manipulation of the microdevices,microdevice-moiety complex, or microdevice-binding partner-moietycomplex includes the directed movement, focusing and trapping of them.The motion of magnetic particles in a magnetic field is termed“magnetophoresis”. Theories and practice of magnetophoresis for cellseparation and other applications may be found in various literatures(e.g., Magnetic Microspheres in Cell Separation, by Kronick, P. L. inMethods of Cell Separation, Volume 3, edited by N. Catsimpoolas, 1980,pages 115-139; Use of magnetic techniques for the isolation of cells, bySafarik I. And Safarikova M., in J. of Chromatography, 1999, Volume722(B), pages 33-53; A fully integrated micromachined magnetic particleseparator, by Ahn C. H. et al., in J. of Microelectromechanical systems,1996, Volume 5, pages 151-157).

The microdevice can further comprise an element that facilitates and/orenables manipulation of the microdevice and/or a moiety/microdevicecomplex or synthesis on the microdevice. Any suitable element that canbe incorporated to the microdevice and that can alter certain propertiesof the microdevice can be used. For example, the element can beelectrically-conductive or dielectrically-polarizable orelectrically-insulating materials to facilitate and/or enablemanipulation by dielectrophoresis force, materials having high or lowacoustic impedance to facilitate and/or enable manipulation by acousticforce, or charged materials to facilitate and/or enable manipulation byelectrostatic force, etc. The element can be a material of certaincomposition, a cell, a cellular organelle, a virus, a microparticle, anaggregate or complex of molecules and an aggregate or complex thereof.In addition, the binding partners disclosed above and disclosed in theco-pending U.S. patent application Ser. No. 09/636,104, filed Aug. 10,2000 can also be used as the element(s) that facilitates and/or enablesmanipulation of the microdevice and/or a moiety/microdevice complex orsynthesis on the microdevice. Non-limiting examples of the elements mayfurther include deposited or other-procedure-produced materials withspecific physical or chemical properties. Metal films made of Au, Cr,Ti, Pt etc are examples of the elements that can be incorporated intothe microdevices and increase electrical conductivity of themicrodevices. Insulating materials such as polystyrene, paralene, orother plastic polymers are also examples of the elements that may beincorporated into the microdevices and reduce electrical conductivity ofthe microdevices.

The element can facilitate and/or enable manipulation of the microdeviceand/or a moiety/microdevice complex by any suitable physical forceincluding the physical forces disclosed in the co-pending U.S. patentapplication Ser. No. 09/636,104, filed Aug. 10, 2000. For instance, adielectrophoresis force, a traveling-wave dielectrophoresis force, anacoustic force such as one effected via a standing-wave acoustic fieldor a traveling-wave acoustic field, an electrostatic force such as oneeffected via a DC electric field, a mechanical force such as fluidicflow force, or an optical radiation force such as one effected via anoptical intensity field generated by laser tweezers, can be used.

Dielectrophoresis refers to the movement of polarized particles, e.g.,microdevices, microdevice-moiety complex, or microdevice-bindingpartner-moiety complex, in a non-uniform AC electrical field. When aparticle is placed in an electrical field, if the dielectric propertiesof the particle and its surrounding medium are different, dielectricpolarization will occur to the particle. Thus, the electrical chargesare induced at the particle/medium interface. If the applied field isnon-uniform, then the interaction between the non-uniform field and theinduced polarization charges will produce a net force acting on theparticle to cause particle motion towards the region of strong or weakfield intensity. The net force acting on the particle is calleddielectrophoretic force and the particle motion is dielectrophoresis.Dielectrophoretic force depends on the dielectric properties of theparticles, particle surrounding medium, the frequency of the appliedelectrical field and the field distribution.

Traveling-wave dielectrophoresis is similar to dielectrophoresis inwhich the traveling-electric field interacts with the field-inducedpolarization and generates electrical forces acting on the particles.Particles, e.g., microdevices, microdevice-moiety complex, ormicrodevice-binding partner-moiety complex, are caused to move eitherwith or against the direction of the traveling field. Traveling-wavedielectrophoretic forces depend on the dielectric properties of theparticles and their suspending medium, the frequency and the magnitudeof the traveling-field. The theory for dielectrophoresis andtraveling-wave dielectrophoresis and the use of dielectrophoresis formanipulation and processing of microparticles may be found in variousliteratures (e.g., “Non-uniform Spatial Distributions of Both theMagnitude and Phase of AC Electric Fields determine DielectrophoreticForces by Wang et al., in Biochim Biophys Acta Vol. 1243, 1995, pages185-194”, “Dielectrophoretic Manipulation of Particles by Wang et al, inIEEE Transaction on Industry Applications, Vol. 33, No. 3, May/June,1997, pages 660-669”, “Electrokinetic behavior of colloidal particles intraveling electric fields: studies using yeast cells by Huang et al, inJ. Phys. D: Appl. Phys., Vol. 26, pages 1528-1535”, “Positioning andmanipulation of cells and microparticles using miniaturized electricfield traps and traveling waves. By Fuhr et al., in Sensors andMaterials. Vol. 7: pages 131-146”, “Dielectrophoretic manipulation ofcells using spiral electrodes by Wang, X-B. et al., in Biophys. J.Volume 72, pages 1887-1899, 1997”, “Separation of human breast cancercells from blood by differential dielectric affinity by Becker et al, inProc. Natl. Acad. Sci., Vol., 92, January 1995, pages 860-864”). Themanipulation of microparticles with dielectrophoresis and traveling wavedielectrophoresis includes concentration/aggregation, trapping,repulsion, linear or other directed motion, levitation, and separationof particles. Particles may be focused, enriched and trapped in specificregions of the electrode reaction chamber. Particles may be separatedinto different subpopulations over a microscopic scale. Particles may betransported over certain distances. The electrical field distributionnecessary for specific particle manipulation depends on the dimensionand geometry of microelectrode structures and may be designed usingdielectrophoresis theory and electrical field simulation methods.

The dielectrophoretic force F_(DEP) _(z) acting on a particle of radiusr subjected to a non-uniform electrical field may be given, under dipoleapproximation, byF _(DEP) _(z) =2πε_(m) r ³χ_(DEP) ∇E _(rms) ² ·{right arrow over (a)}_(z)where E_(rms) is the RMS value of the field strength, ε_(m) is thedielectric permittivity of the medium. χ_(DEP) is the particledielectric polarization factor or dielectrophoresis polarization factor,given, under dipole approximation, by

${\chi_{DEP} = {{Re}\left( \frac{ɛ_{p}^{*} - ɛ_{m}^{*}}{ɛ_{p}^{*} + {2ɛ_{m}^{*}}} \right)}},$“Re” refers to the real part of the “complex number”. The symbol

$ɛ_{x}^{*} = {ɛ_{x} - {j\;\frac{\sigma_{x}}{2\;\pi\; f}}}$is the complex permittivity (of the particle x=p, and the medium x=m).The parameters ε_(p) and σ_(p) are the effective permittivity andconductivity of the particle, respectively. These parameters may befrequency dependent. For example, a typical biological cell will havefrequency dependent, effective conductivity and permittivity, at least,because of cytoplasm membrane polarization.

The above equation for the dielectrophoretic force can also be writtenasF _(DEP) _(z) =2πε_(m) r ³χ_(DEP) V ² p(z){right arrow over (a)} _(z)where p(z) is the square-field distribution for a unit-voltageexcitation (V=1 V) on the electrodes, V is the applied voltage.

There are generally two types of dielectrophoresis, positivedielectrophoresis and negative dielectrophoresis. In positivedielectrophoresis, particles are moved by dielectrophoresis forcestowards the strong field regions. In negative dielectrophoresis,particles are moved by dielectrophoresis forces towards weak fieldregions. Whether particles exhibit positive and negativedielectrophoresis depends on whether the particles are more or lesspolarizable than the surrounding medium.

Traveling-wave DEP force refers to the force that is generated onparticles or molecules due to a traveling-wave electric field. Atraveling-wave electric field is characterized by the non-uniformdistribution of the phase values of AC electric field components.

Here we analyze the traveling-wave DEP force for an ideal traveling-wavefield. The dielectrophoretic force F_(DEP) acting on a particle ofradius r subjected to a traveling-wave electrical field E_(TWD)=Ecos(2π(ft−z/λ₀)){right arrow over (a)}_(x) (i.e., a x-direction field istraveling along the z-direction) is given, under dipole approximation,byF _(TWD)=−2πε_(m) r ³ζ_(TWD) E ² ·{right arrow over (a)} _(z)where E is the magnitude of the field strength, ε_(m) is the dielectricpermittivity of the medium. ζ_(TWD) is the particle polarization factor,given, under dipole approximation, by

${\zeta_{TWD} = {{Im}\left( \frac{ɛ_{p}^{*} - ɛ_{m}^{*}}{ɛ_{p}^{*} + {2ɛ_{m}^{*}}} \right)}},$“Im” refers to the imaginary part of the “complex number”. The symbol

$ɛ_{x}^{*} = {ɛ_{x} - {j\;\frac{\sigma_{x}}{2\pi\; f}}}$is the complex permittivity (of the particle x=p, and the medium x=m).The parameters ε_(p) and σ_(p) are the effective permittivity andconductivity of the particle, respectively. These parameters may befrequency dependent.

Particles such as biological cells having different dielectricproperties (as defined by permittivity and conductivity) will experiencedifferent dielectrophoretic forces. For traveling-wave DEP manipulationof particles (including biological cells), traveling-wave DEP forcesacting on a particle of 10 micron in diameter can vary between 0.01 and10000 pN.

A traveling wave electric field can be established by applyingappropriate AC signals to the microelectrodes appropriately arranged ona chip. For generating a traveling-wave-electric field, it is necessaryto apply at least three types of electrical signals each having adifferent phase value. One method to produce a traveling wave electricfield is to use four phase-quadrature signals (0, 90, 180 and 270degrees) to energize four linear, parallel electrodes patterned on thechip surface. This set of four electrodes forms a basic, repeating unit.Depending on the applications, there may be more than two such unitsthat are located next to each other. This will produce atraveling-electric field in the space above or near the electrodes. Aslong as electrode elements are arranged following certain spatiallysequential orders, applying phase-sequenced signals will result inestablishment of traveling electrical fields in the region close to theelectrodes.

Both dielectrophoresis and traveling-wave dielectrophoresis forcesacting on particles, e.g., microdevices, microdevice-moiety complex, ormicrodevice-binding partner-moiety complex, depend on not only the fielddistributions (e.g., the magnitude, frequency and phase distribution ofelectrical field components; the modulation of the field for magnitudeand/or frequency) but also the dielectric properties of the particlesand the medium in which particles are suspended or placed. Fordielectrophoresis, if particles are more polarizable than the medium(e.g., having larger conductivities and/or permitivities depending onthe applied frequency), particles will experience positivedielectrophoresis forces and be directed towards the strong fieldregions. The particles that are less polarizable than the surroundingmedium will experience negative dielectrophoresis forces and be directedtowards the weak field regions. For traveling wave dielectrophoresis,particles may experience dielectrophoresis forces that drive them in thesame direction as the field is traveling direction or against it,dependent on the polarization factor ζ_(TWD). The following papersprovide basic theories and practices for dielectrophoresis andtraveling-wave-dielectrophoresis: Huang, et al., J. Phys. D: Appl. Phys.26:1528-1535 (1993); Wang, et al., Biochim. Biophys. Acta. 1243:185-194(1995); Wang, et al., IEEE Trans. Ind. Appl. 33:660-669 (1997).

Microdevices, microdevice-moiety complex, or microdevice-bindingpartner-moiety complex, may be manipulated using acoustic forces, i.e.,using acoustic fields. In one case, a standing-wave acoustic field isgenerated by the superimposition of an acoustic wave generated from anacoustic wave source and its reflective wave. Particles in standing-waveacoustic fields experience the so-called acoustic radiation force thatdepends on the acoustic impedance of the particles and their surroundingmedium. Acoustic impedance is the product of the density of the materialand the velocity of acoustic-wave in the material. Particles with higheracoustic impedance than the surrounding medium are directed towards thepressure nodes of the standing wave acoustic field. Particles experiencedifferent acoustic forces in different acoustic field distributions.

One method to generate an acoustic wave source is to use piezoelectricmaterial. These materials, upon applying electrical fields atappropriate frequencies, can generate mechanical vibrations that aretransmitted into the medium surrounding the materials. One type ofpiezoelectric material is piezoelectric ceramics. Microelectrodes may bedeposited on such ceramics to activate the piezoelectric ceramic andthus to produce appropriate acoustic wave fields. Various geometry anddimensions of microelectrodes may be used according to the requirementsof different applications. Reflective walls are needed to generate astanding-wave acoustic field. Acoustic wave fields of variousfrequencies may be applied, i.e., fields at frequencies between kHz andhundred megahertz. In another case, one could use a non-standing waveacoustic field, e.g., a traveling-wave acoustic field. A traveling-waveacoustic field may exert forces on particles (see e.g., see, “Acousticradiation pressure on a compressible sphere, by K. Yoshioka and Y.Kawashima in Acustica, 1955, Vol. 5, pages 167-173”). Particles not onlyexperience forces from acoustic fields directly but also experienceforces due to surrounding fluid because the fluid may be induced to moveby the traveling-wave acoustic field. Using acoustic fields, particlesmay be focussed, concentrated, trapped, levitated and transported in amicrofluidic environment. Another mechanism for producing forces onparticles in an acoustic field is through acoustic-induced fluidconvection. An acoustic field produced in a liquid may induce liquidconvection. Such convection is dependent on the acoustic fielddistribution, properties of the liquid, and the volume and structure ofthe chamber in which the liquid is placed. Such liquid convection willimpose forces on particles placed in the liquid and those forces may beused for manipulating particles. One example where such manipulatingforces may be exploited is for enhancing the mixing of liquids or themixing of particles in a liquid. For the present invention, suchconvection may be used to enhance the mixing of the binding partnerscoupled onto the microdevices with moiety in a suspension and to promotethe interaction between the moiety and the binding partners.

A standing plane wave of ultrasound can be established by applying ACsignals to the piezoelectric transducers. For example, the standing wavespatially varying along the z axis in a fluid can be expressed as:Δp(z)=p ₀ sin(kz)cos(ωt)where Δp is acoustic pressure at z, p₀ is the acoustic pressureamplitude, k is the wave number (2π/λ, where λ is the wavelength), z isthe distance from the pressure node, ω is the angular frequency, and tis the time. According to the theory developed by Yoshioka and Kawashima(see, “Acoustic radiation pressure on a compressible sphere, by K.Yoshioka and Y. Kawashima in Acustica, 1955, Vol. 5, pages 167-173”),the radiation force F_(acoustic) acting on a spherical particle in thestationary standing wave field is given by (see “Studies on particleseparation by acoustic radiation force and electrostatic force by YasudaK. et al. in Jpn. J. Appl. Physics, 1996, Volume 35, pages 3295-3299”)

$F_{acoustic} = {{- \frac{4\pi}{3}}r^{3}k\; E_{acoustic}A\;{\sin\left( {2{kz}} \right)}}$where r is the particle radius, E_(acoustic) is the average acousticenergy density, A is a constant given by

$A = {\frac{{5\rho_{p}} - {2\rho_{m}}}{{2\rho_{p}} + \rho_{m}} - \frac{\gamma_{p}}{\gamma_{m}}}$where ρ_(m) and ρ_(p) are the density of the particle and the medium,γ_(m) and γ_(p) are the compressibility of the particle and medium,respectively. A is termed herein as the acoustic-polarization-factor.

When A>0, the particle moves towards the pressure node (z=0) of thestanding wave.

When A<0, the particle moves away from the pressure node.

Clearly, particles of different density and compressibility willexperience different acoustic-radiation-forces when placed into the samestanding acoustic wave field. For example, the acoustic radiation forceacting on a particle of 10 micron diameter can vary between 0.01 and1000 pN, depending on the established acoustic energy densitydistribution.

Piezoelectric transducers are made from “piezoelectric materials” thatproduce an electric field when exposed to a change in dimension causedby an imposed mechanical force (piezoelectric or generator effect).Conversely, an applied electric field will produce a mechanical stress(electrostrictive or motor effect) in the materials. They transformenergy from mechanical to electrical and vice-versa. The piezoelectriceffect was discovered by Pierre Curie and his brother Jacques in 1880.It is explained by the displacement of ions, causing the electricpolarization of the materials' structural units. When an electric fieldis applied, the ions are displaced by electrostatic forces, resulting inthe mechanical deformation of the whole material.

Microdevices, microdevice-moiety complex, or microdevice-bindingpartner-moiety complex, may be manipulated using DC electric fields. ADC electric field can exert an electrostatic force on charged particles.The force depends on the charge magnitude and polarity of the particlesas well as on the magnitude and direction of the field. The particleswith positive and negative charges may be directed to electrodes withnegative and positive potentials, respectively. By designing amicroelectrode array in a microfluidic device, electric fielddistributions may be appropriately structured and realized. With DCelectric fields, microparticles may be concentrated (enriched), focussedand moved (transported) in a microfluidic device. Proper dielectriccoating may be applied on to DC electrodes to prevent and reduceundesired surface electrochemistry and to protect electrode surfaces.

The electrostatic force F_(E) on a particle in an applied electricalfield E_(z){right arrow over (a)}_(z) can be given byF_(E)=Q_(p)E_(z){right arrow over (a)}_(z)where Q_(p) is the effective electric charge on the particle. Thedirection of the electrostatic force on a charged particle depends onthe polarity of the particle charge as well as the direction of theapplied field.

Thermal convection forces refer to the forces acting on particles, e.g.,microdevices, microdevice-moiety complex, or microdevice-bindingpartner-moiety complex, due to the fluid-convection (liquid-convection)that is induced by a thermal gradient in the fluid. Thermal diffusion inthe fluid drives the fluid towards thermal equilibrium. This causes afluid convection. In addition, the density of aqueous solutions tends todecrease with increasing temperature. Such density differences are alsonot stable within a fluid resulting in convection. Thermal convectionmay be used to facilitate liquid mixing. Directed thermal convection mayact as an active force.

Thermal gradient distributions may be established within a chip-basedchamber where heating and/or cooling elements may be incorporated intothe chip structure. A heating element may be a simple joule-heatingresistor coil. Such a coil could be microfabricated onto the chip. As anexample, consider a coil having a resistance of 10 ohm. Applying 0.2 Athrough the coil would result in 0.4 W joule heating-power. When thecoil is located in an area <100 micron², this is an effective way ofheat generation. Similarly, a cooling element may be a Peltier elementthat could draw heat upon applying electric potentials.

As an exemplary embodiment, the microdevices of the present inventionmay be used on a chip that incorporates an array of individuallyaddressable heating elements. These heating elements may be positionedor structurally arranged in certain order so that when each, some, orall of the elements are activated, thermal gradient distributions willbe established to produce thermal convection. For example, if oneheating element is activated, temperature increases in the liquid in theneighborhood of that element will induce fluid convection. In anotherexemplary embodiment, the chip may comprise multiple, interconnectedheating units so that these units can be turned on or off in asynchronized order. Yet, in another example, the chip may comprise onlyone heating element that can be energized to produce heat and inducethermal convection in the liquid fluid.

Other physical forces may be applied. For example, mechanical forces,e.g., fluidic flow forces, may be used to transport microparticles,e.g., microdevices, microdevice-moiety complex, or microdevice-bindingpartner-moiety complex. Optical radiation forces as exploited in “lasertweezers” may be used to focus, trap, levitate and manipulatemicroparticles. The optical radiation forces are the so-calledgradient-forces when a material (e.g., a microparticle) with arefractive index different from that of the surrounding medium is placedin a light gradient. As light passes through a polarizable material, itinduces fluctuating dipoles. These dipoles interact with theelectromagnetic field gradient, resulting in a force directed towardsthe brighter region of the light if the material has a refractive indexlarger than that of the surrounding medium. Conversely, an object with arefractive index lower than the surrounding medium experiences a forcedrawing it towards the darker region. The theory and practice of “lasertweezers” for various biological application are described in variousliteratures (e.g., “Making light work with optical tweezers, by Block S.M., in Nature, 1992, Volume 360, pages 493-496”; “Forces of asingle-beam gradient laser trap on a dielectric sphere in the ray opticsregime, by Ashkin, A., in Biophys. J., 1992, Volume 61, pages 569-582”;“Laser trapping in cell biology, by Wright et al., in IEEE J. of QuantumElectronics, 1990, Volume 26, pages 2148-2157”; “Laser manipulation ofatoms and particles, by Chu S. in Science, 1991, Volume 253, pages861-866”). The light field distribution and/or light intensitydistribution may be produced with built-in optical elements and arrayson a chip and external optical signal sources, or may be produced withbuilt-in electro-optical elements and arrays on a chip and the externalstructures are electrical signal sources. In the former case, when thelight produced by the optical signal sources passes through the built-inoptical elements and arrays, light is processed by these elements/arraysthrough, e.g., reflection, focusing, interference, etc. Optical fielddistributions are generated in the regions around the chip. In thelatter case, when the electrical signals from the external electricalsignal sources are applied to the built-in electro-optical elements andarrays, light is produced from these elements and arrays and opticalfields are generated in the regions around the chip.

Although the microdevices can comprise a single element that canfacilitate and/or enable manipulation of the microdevice by one type ofphysical forces or synthesis on the microdevice, they may also be usedin high throughput analysis and preferably comprise a plurality ofelements, each of the elements facilitates and/or enables manipulationof the microdevice and/or the moiety/microdevice complex by a differentphysical force. For example, the element can be a conductive orinsulating material for manipulation by a dielectrophoresis force, amaterial having high or low acoustic impedance for manipulation byacoustic force, and/or a charged material for manipulation by aelectrostatic force, etc.

In a preferred embodiment, the microdevice comprises a binding partnerthat is capable of binding or specifically binding to a moiety to beisolated, manipulated, detected or synthesized and an element thatfacilitates and/or enables manipulation of the microdevice and/or themoiety/microdevice complex. More preferably, the microdevice(s)comprises a plurality of binding partners, each of the binding partnersis capable of binding or specifically binding to a different moiety tobe isolated, manipulated, detected or synthesized and a plurality of theelements, each of the elements facilitates and/or enables manipulationof the microdevice and/or the moiety/microdevice complex by a differentphysical force.

The microdevice can further comprise a detectable marker or a moleculartag. Exemplary detectable markers include dyes, radioactive substancesand fluorescent substances. Exemplary detectable molecular tags includenucleic acid, oligonucleotide, protein and peptide sequences.

In a specific embodiment, the present microdevice has a thin rectangularshape and has a major axis (length) to minor axis (width) ratio of atleast about 1.2, and preferably at least about 1.5, and has a thickness(height) smaller than both major axis and minor axis. In anotherspecific embodiment, the present microdevice comprises at least tworectangular-shaped strips (or bars) or near-rectangular-shaped strips(or bars) of the paramagnetic substance. Preferably, at least two strips(or bars) of the paramagnetic substance are separated and located oneach side of the microdevice along the major axis of the microdevice.More preferably, a metal film is processed to have a photorecognizablepattern that is located between the at least two strips (or bars) of theparamagnetic substances. More preferably, the metal film comprisesaluminum. Also more preferably, the present microdevice has unequalnumber of the paramagnetic substance strip(s) (or bars) on each sidealong the major axis of the microdevice. In still another specificembodiment, the present microdevice comprises two strips (or bars) ofthe paramagnetic substance along the major axis of the microdevice.Preferably, the two strips (or bars) of the paramagnetic substance havefingers on both ends. In yet another specific embodiment, theparamagnetic substance in the present microdevice forms a strip (or bar)along the major axis of the microdevice and said strip (or bar) hasfingers on both ends.

In another aspect, the present invention is directed to a system forforming a microdevice array, which system comprises: a) a plurality ofmicrodevices, each of the microdevices comprising a magnetizablesubstance and a photorecognizable coding pattern, wherein saidmicrodevices have a preferential axis of magnetization; and b) amicrochannel array comprising a plurality of microchannels, saidmicrochannels are sufficiently wide to permit rotation of saidmicrodevices within said microchannels but sufficiently narrow toprevent said microdevices from forming a chain when the major axis ofsaid microdevices is substantially perpendicular to the major axis ofsaid microchannels wherein the said microdevices are subjected to anapplied magnetic field.

In preferred embodiments, the microdevices are manipulated to be “flat”or “substantially flat” in the microchannels so that thephotorecognizable patterns on the microdevices can be optically detectedor analyzed via the optical means in the direction substantially-normalto the plane defined by the microchannel length and width. In preferredembodiments, the height of the microchannels and/or the constraint onthe microdevices by a magnetic field should be adjusted to prevent themicrodevices from standing up within the microchannels. In a specificembodiment, the height of the microchannels is less than about 70% ofthe major axis of the microdevices.

The microchannel array can further comprise a staging area or loadingarea where the microdevices can be introduced into and/or an output areaor an outlet channel where the microdevices may be removed from themicrochannel array. The microchannel array can also further comprise amagnetic field generating means capable of generating a magnetic fieldsuitable for manipulating the microdevices into, within and/or out ofthe microchannel array, or rotating the microdevices within themicrochannel array. Any suitable magnetic field generating means can beused. For example, the magnetic field generating means can comprise apermanent magnet, a mobile permanent magnet, an electromagnetic unit, aferromagnetic material or a microelectromagnetic unit. The magneticfield generating means can be located at any suitable location, e.g.,below, within, above and/or near the microchannel array.

C. Methods for Forming a Microdevice Array

In still another aspect, the present invention is directed to a methodfor forming a microdevice array, which method comprises: a) providing aplurality of microdevices, each of the microdevices comprising amagnetizable substance and a photorecognizable coding pattern, whereinsaid microdevices have a preferential axis of magnetization; b)providing a microchannel array comprising a plurality of microchannels,said microchannels are sufficiently wide to permit rotation of saidmicrodevices within said microchannels but sufficiently narrow toprevent said microdevices from forming a chain when the major axis ofsaid microdevices is substantially perpendicular to the major axis ofsaid microchannels wherein said microdevices are subjected to an appliedmagnetic field; c) introducing said plurality of microdevices into saidplurality of microchannels; and d) rotating said microdevices withinsaid microchannels by a magnetic force, whereby the combined effect ofsaid magnetic force and said preferential axis of magnetization of saidmicrodevices substantially separates said microdevices from each other.

In preferred embodiments, the microdevices are manipulated to be “flat”or “substantially flat” in the microchannels so that thephotorecognizable patterns on the microdevices can be optically detectedor analyzed via the optical means in the direction substantially-normalto the plane defined by the microchannel length and width. In preferredembodiments, the height of the microchannels and/or the constraint onthe microdevices by a magnetic field should be adjusted to prevent themicrodevices from standing up within the microchannels. In a specificembodiment, the height of the microchannels is less than about 70% ofthe major axis of the microdevices.

The microdevices can be introduced into the microchannels by anysuitable force. For example, the microdevices can be introduced into themicrochannels by a magnetic force, a fluidic force or a combinationthereof. There are multiple methods for introducing or loading themicrodevices into the channels. In one example, the microdevices are inthe form of the MicroDisks, which have two major surfaces and a smalldimension (small thickness) between the two major surfaces. MicroDisksare placed in the loading area near the inlet to the microchannels orchannels. A small Neodymium magnet at the outlet end of the channel isused to draw the MicroDisks into the channel. The magnet is rotated tofacilitate movement of the MicroDisks into the channels. In oneexperiment, the MicroDisk's major surfaces are of dimensions of 90 μm by70 μm and the MicroDisks are several μm thick. Using the above-describedprocedure, it was possible to completely fill five 2 cm long channels(channel widths ranging from 120-160μ) with 90×70μ MicroDisks(containing magnetic strips (or bars) with the “3-finger” pattern) inless than 3 minutes. The length, width and height directions of themagnetic strips or bars correspond, respectively to, the length, widthand height directions of the MicroDisks. Since the rate-limiting step inthe loading or filling process is the MicroDisks moving along the lengthof the channel, the number of channels can be increased withoutsignificantly affecting loading or filling time, e.g., two hundred 2 cmlong channels can be filled with about 50,000 MicroDisks within a3-minute time-period using this procedure. Channels loaded in thismanner may be overloaded such that when the direction of the appliedexternal magnetic field is perpendicular to the channel the“perpendicularly arrayed” MicroDisks will be overlapping. Overlaps canbe relieved by alternating the direction of the applied externalmagnetic field between perpendicular and parallel several times. Thiscauses the “chains” to lengthen. In this example, preferably, MicroDisksare introduced into the channels or microchannels so that the heightdirection of the MicroDisks is substantially aligned with the heightdirection of the microchannels or channels. Preferably, MicroDisks thathave been loaded and/or arrayed into the channels or microchannels arelying flat on the surface of the microchannels.

In another example, the microdevices are in the form of the MicroDisks,which have two major surfaces and a small dimension (small thickness)between the two major surfaces. MicroDisks are loaded into themicrochannels or channels exactly as described in above example with theaddition of a steady flow-rate of liquid through the channels toincrease the efficiency of channel loading. In this example, preferably,MicroDisks are introduced into the channels or microchannels so that theheight direction of the MicroDisks is substantially aligned with theheight direction of the microchannels or channels. Preferably,MicroDisks that have been loaded and/or arrayed into the channels ormicrochannels are lying flat on the surface of the microchannels.

In still another example, the microdevices are in the form of theMicroDisks, which have two major surfaces and a small dimension (smallthickness) between the two major surfaces. The MicroDisks comprisesmagnetic strips or bars, whose length, width and height directionscorrespond, respectively, to the length, width and height directions ofthe MicroDisks. MicroDisks are placed in the loading area near the inletto the channels. A large Neodymium magnet at the outlet end of thechannel is used to draw the MicroDisks into the channel. The magnetfield from this magnet is perpendicular to the channels. A smallNeodymium magnet is placed above or below the inlet to the channels androtated to facilitate movement of the MicroDisks into the channels. Asteady flow-rate of liquid through the channels increases the efficiencyof channel loading. MicroDisks are loaded in their final“perpendicularly arrayed” form (preferential axis of magnetizationperpendicular to the long (or major) axis of the channel), minimizingchannel overloading and providing a more uniform arraying pattern. Themethod of loading or arraying the MicroDisks in this example will resultin the magnetic bars within or on the MicroDisks being perpendicular tothe channel after the MicroDisks are loaded into the channels ormicrochannels, i.e. the length direction of the magnetic strips or bars(i.e. the length direction of the MicroDisks) will be normal orsubstantially normal to the length direction of the microchannels. Inthis example, preferably, MicroDisks are introduced into the channels ormicrochannels so that the height direction of the MicroDisks issubstantially aligned with the height direction of the microchannels orchannels. Preferably, MicroDisks that have been loaded and/or arrayedinto the channels or microchannels are lying flat on the surface of themicrochannels.

The microdevices or MicroDisks can be introduced into the microchannelsat any suitable angle. For example, the microdevices can be introducedinto the microchannels by a magnetic field at a direction such that theangle between the major axis of the microdevice and the microchannel isabout less than 45 degrees. The direction of the magnetic field willaffect the orientation of the microdevices or MicroDisks and affect thedirection of the major axis of the microdevice. Normally, when themicrodevices can freely rotate or re-orientate, the preferential axis ofmagnetization is substantially aligned with the applied magnetic field.For microdevice or MicroDisk whose preferential axis of magnetization isthe same as, or substantially aligned with, the major axis of themicrodevice, then the major axis of the microdevice is substantiallyaligned with the applied magnetic field. It is thus possible tointroduce the microdevices into the microchannels by a magnetic field atappropriate directions so that the major axis of the microdevice isangled with respect to the length direction of the microchannels atdegrees less than 45 degree. Preferably, the microdevices are introducedinto the microchannels by a magnetic field at a direction such that theangle between the major axis of the microdevice and the microchannel isabout less than 40, 35, 30, 25, 20, 15, 10, 5 or 0 degrees.

The present method can further comprise a step of breaking a chainformed among the microdevices prior to or concurrent with introducingthe microdevices into the microchannels. This can be accomplished by anysuitable methods, e.g., rotating the direction of magnetic field betweenthe major and minor axis of the microdevices.

Preferably, after microdevices or MicroDisks are loaded and/or filledinto the channels or microchannels, microdevices or MicroDisks are lyingflat on the surface of the microchannels. The microdevices or MicroDiskscan be rotated within the microchannels for any suitable degreesprovided that the rotation is sufficient to substantially separate themicrodevices or MicroDisks from each other. The separation can beachieved by a single rotation of a larger degree or by multiplerotations for smaller degrees. Preferably, the microdevices orMicroDisks are rotated at least 45 degrees. More preferably, themicrodevices or MicroDisks are rotated 90 degrees.

In a specific embodiment, at least one of the microdevices binds to amoiety and the method is used to manipulate said moiety. In anotherspecific embodiment, a plurality of the microdevices bind to a pluralityof moieties and the method is used to manipulate said plurality ofmoieties. The present method can be used for any suitable manipulationof a moiety, e.g., transportation, focusing, enrichment, concentration,aggregation, trapping, repulsion, levitation, separation, fractionation,isolation and linear or other directed motion of the moiety. In stillanother specific embodiment, the present method can further comprise astep of assessing the identity of the manipulated moiety byphotoanalysis of the photorecognizable coding pattern on the microdeviceto which the moiety binds. Assessment of the identity of the manipulatedmoiety may involve obtaining an index, ratio, percentage, visual orother value indicative of the identity of the manipulated moiety. Instill another specific embodiment, the present method can furthercomprise a step of assessing the quantity of the manipulated moiety byfurther quantitative means for analyzing the amount of the manipulatedmoiety on the microdevice. The assessment of the quantity of themanipulated moiety may involve obtaining a number, an index, or othervalue indicative of the amount or quantity or the concentration of themanipulated moiety. In yet another specific embodiment, the presentmethod can further comprise a step of collecting the microdevice towhich the moiety binds through an outlet channel. The present method canfurther comprise a step of recovering the moiety from the collectedmicrodevice.

In yet another aspect, the present invention is directed to a method forforming a microdevice array, which method comprises: a) providing aplurality of microdevices, each of the microdevices comprising amagnetizable substance and a photorecognizable coding pattern, whereinsaid microdevices have a preferential axis of magnetization, on asurface suitable for rotation of said microdevices; and b) rotating saidmicrodevices on said surface by a magnetic force, whereby the combinedeffect of said magnetic force and said preferential axis ofmagnetization of said microdevices substantially separates saidmicrodevices from each other.

In yet another aspect, the present invention is directed to a method forforming a microdevice array, which method comprises: a) providing aplurality of the microdevices, each of the microdevices comprising amagnetizable substance and a photorecognizable coding pattern, andhaving a preferential axis of magnetization; b) introducing saidplurality of microdevices onto a surface; and rotating said microdevicesby a magnetic force to form chains and clusters, whereby the combinedeffect of said magnetic force and said preferential axis ofmagnetization of said microdevices substantially separates themicrodevices from each other. In an embodiment of the arraying method,the microdevices are introduced onto the surface in a liquid suspension.The microdevice suspension can be added to the surface by a variety ofmethods, such as via micropieppetting, or pumping into the microchannelsthat are formed on the surface. In another embodiment of the methods,the surface may comprise grooves with width dimensions substantiallynarrower than that of the microdevice. After the microdevice are arrayedon the surface, the liquid in which the microdevices are suspended maybe removed via the grooves on the surfaces by various methods such assuction or pumping out.

The present methods can be used for analyzing, isolating, manipulatingor detecting any types of moieties when the moieties are involved incertain processes, such as physical, chemical, biological, biophysicalor biochemical processes, etc., in a chip format or non-chip format.Moieties can be cells, cellular organelles, viruses, molecules or anaggregate or complex thereof. Moieties can be pure substances or canexist in a mixture of substances wherein the target moiety is only oneof the substances in the mixture. For example, cancer cells in the bloodfrom leukemia patients, cancer cells in the solid tissues from patientswith solid tumors and fetal cells in maternal blood from pregnant womencan be the moieties to be isolated, manipulated or detected. Similarly,various blood cells such as red and white blood cells in the blood canbe the moieties to be isolated, manipulated or detected. DNA molecules,mRNA molecules, certain types of protein molecules, or all proteinmolecules from a cell lysate can be moieties to be isolated, manipulatedor detected.

Non-limiting examples of cells include animal cells, plant cells, fungi,bacteria, recombinant cells or cultured cells. Animal, plant cells,fungus, bacterium cells to be isolated, manipulated or detected can bederived from any genus or subgenus of the Animalia, Plantae, fungus orbacterium kingdom. Cells derived from any genus or subgenus of ciliates,cellular slime molds, flagellates and microsporidia can also beisolated, manipulated or detected. Cells derived from birds such aschickens, vertebrates such fish and mammals such as mice, rats, rabbits,cats, dogs, pigs, cows, ox, sheep, goats, horses, monkeys and othernon-human primates, and humans can be isolated, manipulated or detectedby the present methods.

For animal cells, cells derived from a particular tissue or organ can beisolated, manipulated or detected. For example, connective, epithelium,muscle or nerve tissue cells can be isolated, manipulated or detected.Similarly, cells derived from an accessory organ of the eye,annulospiral organ, auditory organ, Chievitz organ, circumventricularorgan, Corti organ, critical organ, enamel organ, end organ, externalfemale genital organ, external male genital organ, floating organ,flower-spray organ of Ruffini, genital organ, Golgi tendon organ,gustatory organ, organ of hearing, internal female genital organ,internal male genital organ, intromittent organ, Jacobson organ,neurohemal organ, neurotendinous organ, olfactory organ, otolithicorgan, ptotic organ, organ of Rosenmüller, sense organ, organ of smell,spiral organ, subcommissural organ, subformical organ, supernumeraryorgan, tactile organ, target organ, organ of taste, organ of touch,urinary organ, vascular organ of lamina terminalis, vestibular organ,vestibulocochlear organ, vestigial organ, organ of vision, visual organ,vomeronasal organ, wandering organ, Weber organ and organ of Zuckerkandlcan be isolated, manipulated or detected. Preferably, cells derived froman internal animal organ such as brain, lung, liver, spleen, bonemarrow, thymus, heart, lymph, blood, bone, cartilage, pancreas, kidney,gall bladder, stomach, intestine, testis, ovary, uterus, rectum, nervoussystem, gland, internal blood vessels, etc can be isolated, manipulatedor detected. Further, cells derived from any plants, fingi such asyeasts, bacteria such as eubacteria or archaebacteria can be isolated,manipulated or detected. Recombinant cells derived from any eucaryoticor prokaryotic sources such as animal, plant, fungus or bacterium cellscan also be isolated, manipulated or detected. Cells from various typesof body fluid such as blood, urine, saliva, bone marrow, sperm or otherascitic fluids, and subfractions thereof, e.g., serum or plasma, canalso be isolated, manipulated or detected.

Isolatable, manipulatable or detectable cellular organelles includenucleus, mitochondria, chloroplasts, ribosomes, ERs, Golgi apparatuses,lysosomes, proteasomes, secretory vesicles, vacuoles or microsomes.Isolatable, manipulatable or detectable viruses include intact virusesor any viral structures, e.g., viral particles, in the virus life cyclethat can be derived from viruses such as Class I viruses, Class IIviruses, Class III viruses, Class IV viruses, Class V viruses or ClassVI viruses.

Isolatable, manipulatable or detectable molecules can be inorganicmolecules such as ions, organic molecules or a complex thereof.Non-limiting examples of ions include sodium, potassium, magnesium,calcium, chlorine, iron, copper, zinc, manganese, cobalt, iodine,molybdenum, vanadium, nickel, chromium, fluorine, silicon, tin, boron orarsenic ions. Non-limiting examples of organic molecules include aminoacids, peptides, proteins, nucleosides, nucleotides, oligonucleotides,nucleic acids, vitamins, monosaccharides, oligosaccharides,carbohydrates, lipids or a complex thereof.

Any amino acids can be isolated, manipulated or detected by the presentmethods. For example, a D- and a L-amino-acid can be isolated,manipulated or detected. In addition, any building blocks of naturallyoccurring peptides and proteins including Ala (A), Arg (R), Asn (N), Asp(D), Cys (C), GIn (O), Glu (E), Gly (G), His (H), Ile (I), Leu (L), Lys(K), Met (M), Phe (F), Pro (P) Ser (S), Thr (T), Trp (W), Tyr (Y) andVal (V) can be isolated, manipulated or detected.

Any proteins or peptides can be isolated, manipulated or detected by thepresent methods. For example, membrane proteins such as receptorproteins on cell membranes, enzymes, transport proteins such as ionchannels and pumps, nutrient or storage proteins, contractile or motileproteins such as actins and myosins, structural proteins, defenseprotein or regulatory proteins such as antibodies, hormones and growthfactors can be isolated, manipulated or detected. Proteineous orpeptidic antigens can also be isolated, manipulated or detected.

Any nucleic acids, including single-, double and triple-stranded nucleicacids, can be isolated, manipulated or detected by the present methods.Examples of such nucleic acids include DNA, such as A-, B- or Z-formDNA, and RNA such as mRNA, tRNA and rRNA.

Any nucleosides can be isolated, manipulated or detected by the presentmethods. Examples of such nucleosides include adenosine, guanosine,cytidine, thymidine and uridine. Any nucleotides can be isolated,manipulated or detected by the present methods. Examples of suchnucleotides include AMP, GMP, CMP, UMP, ADP, GDP, CDP, UDP, ATP, GTP,CTP, UTP, dAMP, dGMP, dCMP, dTMP, dADP, dGDP, dCDP, dTDP, dATP, dGTP,dCTP and dTTP.

Any vitamins can be isolated, manipulated or detected by the presentmethods. For example, water-soluble vitamins such as thiamine,riboflavin, nicotinic acid, pantothenic acid, pyridoxine, biotin,folate, vitamin B₁₂ and ascorbic acid can be isolated, manipulated ordetected. Similarly, fat-soluble vitamins such as vitamin A, vitamin D,vitamin E, and vitamin K can be isolated, manipulated or detected.

Any monosaccharides, whether D- or L-monosaccharides and whether aldosesor ketoses, can be isolated, manipulated or detected by the presentmethods. Examples of monosaccharides include triose such asglyceraldehyde, tetroses such as erythrose and threose, pentoses such asribose, arabinose, xylose, lyxose and ribulose, hexoses such as allose,altrose, glucose, mannose, gulose, idose, galactose, talose and fructoseand heptose such as sedoheptulose.

Any lipids can be isolated, manipulated or detected by the presentmethods. Examples of lipids include triacylglycerols such as tristearin,tripalmitin and triolein, waxes, phosphoglycerides such asphosphatidylethanolamine, phosphatidylcholine, phosphatidylserine,phosphatidylinositol and cardiolipin, sphingolipids such assphingomyelin, cerebrosides and gangliosides, sterols such ascholesterol and stigmasterol and sterol fatty acid esters. The fattyacids can be saturated fatty acids such as lauric acid, myristic acid,palmitic acid, stearic acid, arachidic acid and lignoceric acid, or canbe unsaturated fatty acids such as palmitoleic acid, oleic acid,linoleic acid, linolenic acid and arachidonic acid.

D. Methods for Synthesizing a Library and Uses Thereof

In another aspect, the present invention is directed to a method forsynthesizing a random library, which method comprises: a) providing aplurality of microdevices, each of said microdevices comprises amagnetizable substance and a unique photorecognizable coding pattern,wherein each of said microdevices has a preferential axis ofmagnetization and wherein said unique photorecognizable coding patternon each of said microdevices corresponds to an entity to be synthesizedon each of the said microdevices; and b) synthesizing said entities onsaid microdevices, wherein said microdevices are identified after eachsynthesis cycle according to said unique photorecognizable codingpatterns, whereby a library is synthesized, wherein each of saidmicrodevices contains an entity that corresponds to said uniquephotorecognizable coding pattern on each of the said microdevices andthe sum of said microdevices collectively contains a plurality ofentities. A library that is synthesized according to the above method isalso provided.

In yet another aspect, the present invention is directed to a method forsynthesizing a library of predetermined sequence, which methodcomprises: a) providing a plurality of microdevices, each of saidmicrodevices comprises a magnetizable substance and a photorecognizablecoding pattern, wherein said microdevices have a preferential axis ofmagnetization and wherein said photorecognizable coding patterncorresponds to an entity to be synthesized on said microdevice; and b)synthesizing said entities on said microdevices, wherein saidmicrodevices are sorted after each synthesis cycle according to saidphotorecognizable coding patterns, whereby a library is synthesized,wherein each of said microdevices contains an entity that corresponds toa photorecognizable coding pattern on said microdevice and the sum ofsaid microdevices collectively contains a plurality of entities that ispredetermined before the library synthesis. A library that issynthesized according to the above method is also provided.

The microdevices can be sorted by any suitable methods. For example, themicrodevices can be sorted through a microchannel array comprising aplurality of microchannels, said microchannels are sufficiently wide topermit rotation of said microdevices within said microchannels butsufficiently narrow to prevent said microdevices from forming a chainwhen the major axis of said microdevices is substantially perpendicularto the major axis of said microchannels, via a combined effect of amagnetic force and the preferential axis of magnetization of themicrodevices that substantially separates the microdevices from eachother. The height of the microchannels and/or the constraint on themicrodevices by a magnetic field should be adjusted to prevent themicrodevices from standing up within the microchannels. In a specificembodiment, the height of the microchannels is about less than 70% ofthe major axis of the microdevices. After the microdevices are arrayedinto the microchannels, photoanalysis of microdevices is performed todetermine photorecognizable coding (or encoding) pattern of individualmicrodevice. A method that can handle individual microdevice is used tomanipulate individual microdevice and to sort them to differentregions/locations/reaction chambers according to their photorecognizablepattern. For example, a microelectromagnetic needle that can generatemagnetic field at a fine tip-end of the needle can be used to pick upindividual microdevice from their arrayed channels (in this case, thechannels have to be open on the top side) and move and send/dispenseindividual microdevices to different locations/regions/reactionchambers. In another example, microdevices are moved out from themicrochannels by, e.g., a combination of magnetic forces and fluidicforces, and at the outlet region of the channel, microdevices can besent to different locations by the control of magnetic forces and/orfluidic forces.

Sorting can also be accomplished through the use of magnetic force tospecifically capture desired grouping of microdevices after each step ofthe synthesis and deposit them into the appropriate reaction vessel.Microdevices can be arrayed using a photoresist to form either the topor bottom surface of the arraying chamber. When exposed to light of theappropriate wavelength the photoresist in the illuminated regions can bedissolved exposing the Microdevices in those locations and allowing themto be removed by magnetic force. A programmable digital micromirrorarray (e.g. “Maskless fabrication of light-directed oligonucleotidesmicroarrays using a digital micromirror array” by Singh-Gasson et al.Nature Biotechnology, 17:974-978 (1999)) or similar maskless arraysynthesizer device could be used to direct the light.

An alternative and preferred method of sorting utilizing magnetic forceis to use sorting channels. As discussed above, microdevices having apreferential axis of magnetization when arrayed in a channel in thepresence of a magnetic field will align and separate due to repulsivemagnetic force and can be drawn through liquid filled channels in a“perpendicularly arrayed” form (preferential axis of magnetizationperpendicular to the long axis of the channel). A sufficient increase insurface tension will prevent movement of the microdevice. Such an effectcan be generated by creating an appropriate liquid-liquid (immiscibleliquids such as hexane and water) or gas-liquid interface. For exampleconsider an arraying channel separated from a series of sorting channelsby a microvalve (the design, manufacture, and use of such valves arewell known to those practiced in the art). Through an appropriatelypositioned orifice near the end of the arraying channel a bubble can beintroduced between the final and the penultimate microdevice. Opening ofthe valve and application of a magnetic force will result in only thefinal microdevice being drawn through the channel into the sortingchannels, others microdevices will remain trapped behind the bubble. Thevalve is then closed and the single disk in the sorting channel can bedirected using magnetic and/or fluidic force to and/or other physicalforce (e.g. dielectrophoresis force) the appropriate reaction vessel.Application of fluidic force (pumping liquid) drives the bubble outthrough an appropriately placed outlet at the end of the channelallowing the microdevices to advance and the sorting process is thenrepeated. The size of orifices for gas delivery and removal must besignificantly smaller than the microdevices. An alternative andpotentially more rapid system would be to introduce bubbles between allof the microdevices within a channel and to adjust the magnetic fieldand fluidic force such that the microdevices move in a segmented fashionthrough the channel. This is analogous to the segmented fluid flowapproach widely used by Technicon International, Ltd. to prevent peakbroadening (e.g., U.S. Pat. Nos. 2,797,149 and 3,109,713). A thirdparameter in addition to magnetic and fluidic force which can beadjusted to insure smooth segmented flow of microdevices is the surfacetension of the liquid(s) which can be regulated by the use theappropriate solvents or additives (e.g. surfactants). The ability toalter surface tension by choice of solvents is known to anyone trainedin the art.

In another example, microdevices can be sorted using the apparatuses(i.e.: particle switches) that can switch and manipulate particles. U.S.patent application Ser. No. 09/678,263, filed on Oct. 3, 2000, titled“Apparatus for switching and manipulating particles and methods of usethereof” describe several types of devices and apparatuses forswitching, sorting and manipulating particle. The patent applicationSer. No. 09/678,263 is incorporated by reference in its entirety. Thedevices and apparatuses and the methods of their use can be applied forsorting microdevices of present invention. For example, traveling-wavedielectrophoresis can be used as a mechanism for sorting particles via aparticle-switching device. The particle switching device comprises atleast three sets of electrodes which are electrically independent fromeach other. The three or more sets of electrodes are capable ofgenerating respective traveling-wave dielectrophoresis (twDEP) forces onparticles to move the particles along respective branches when theelectrodes in each set of electrodes are connected to out-of-phasesignals, and said branches are interconnected at a common junction topermit the twDEP forces to route particles from one of the branches toanother of the branches. The end (other than the common junction of thebranches) of each branch may be used for the inlet (input) and/or outlet(output) ports. Thus, in this example, the particle sorting device hasat least three inlet (input)/outlets (outputs). Consider an examplewhere the particle sorting device has one inlet and two outlet ports.Microdevices of the present invention can be fed into the inlet port andthen transported along the branches within the particle sorting deviceto be outputted in one of the two outlets, depending on the electricalvoltage signals applied to the electrodes. More importantly, for a givenmicrodevice of the present invention, it is possible to first performphoto-analysis to determine the photorecognizable coding pattern on themicrodevice and then according to its coding pattern, appropriateelectrical signals can be applied to the electrodes within the particlesorting device so that the microdevice can be transported and sorted toone of the two outlet ports. An array of such particle sorting devicescan be used for sorting microdevices into more than two outlet ports (ormore than two output points/positions). Examples of such multipleparticle-sorting-device used in an array format are also disclosed inthe U.S. patent application Ser. No. 09/678,263, which is incorporatedby reference in its entirety.

Microdevices can also be sorted using a flow system, which has one inletport and multiple outlet ports. The flow system can transportmicrodevices from the inlet port to any one of multiple outlet ports.Each microdevice can be flown through an optical decoder (in the flowsystem), which can identify the photorecognizable coding pattern of themicrodevice, and is then directed to different outlet ports according tothe identified coding pattern on the microdevice by changing the fluidflow patterns in the flow system.

Any other sorting method that can sort microdevices according to theirphotorecognizable coding patterns can be used.

Any number of suitable entity(ies) can be synthesized on a singlemicrodevice. For example, a single entity or a plurality of entities canbe synthesized on a single microdevice. Preferably, a single entity issynthesized on a single microdevice.

The present method can be used to synthesize any kind of library. Forexample, the synthesized entities can be peptides, proteins,oligonucleotides, nucleic acids, vitamins, oligosaccharides,carbohydrates, lipids, small molecules, or a complex or combinationthereof. Preferably, the synthesized library comprises a defined set ofentities that are involved in a biological pathway, belongs to a groupof entities with identical or similar biological function, expressed ina stage of cell cycle, expressed in a cell type, expressed in a tissuetype, expressed in an organ type, expressed in a developmental stage,entities whose expression and/or activity are altered in a disease ordisorder type or stage, or entities whose expression and/or activity arealtered by drug or other treatments.

In a specific embodiment, the synthesized library comprises a definedset of nucleic acid, e.g., DNA or RNA, fragments such as a defined setof nucleic acid fragments that cover an entire genome, e.g., the entirehuman genome sequence. Preferably, each of the nucleic acid fragments inthe synthesized library comprises at least 2, 3, 5, 10, 15, 20, 25, 50,75, 100, 200, or 500 nucleotides.

In another specific embodiment, the synthesized library comprises adefined set of protein or peptide fragments such as a defined set ofprotein or peptide fragments that cover protein or peptide sequencesencoded by an entire genome, e.g., the entire human genome sequence.Preferably, each of the protein or peptide fragments in the synthesizedlibrary comprises at least 2, 3, 5, 10, 15, 20, 25, 50, 75, 100, 150,200, 300, 400 or 500 amino acid residues.

In still another specific embodiment, a library that is synthesizedaccording to the above-described method is provided.

E. Preferred Embodiments

In one specific embodiment, the present invention is directed toward amethod for arraying microdevices (or MicroDisks) in predeterminedgeometries using magnetic forces. A MicroDisk is a microfabricatedparticle ranging in size from 1-1000μ on a side and containing one ormore strips or bars of magnetic material. These bars must have theproperty of having a preferential axis of magnetization. Such a propertyis a consequence of the physical geometry of the magnetic material and,typically, will consist of a thin film (generally less than 1μ) barhaving a length to width ratio of greater than 3. Typically, thepreferential axis of magnetization of a bar is its major axis. Anexample of a MicroDisk containing two magnetic strips or bars is shownin FIG. 1. For example in the presence of a magnetic field as indicatedby the arrow, the MicroDisks will orient or rotate so that thepreferential axis of magnetization will be parallel or substantiallyparallel to the field. For such MicroDisks, the preferential axis ofmagnetization is aligned with its major axis, or is its major axis. Ifnot spatially constrained, MicroDisks will form chains and clusters asshown in FIG. 2 (the arrow indicating the direction of applied magneticfield). Chains may be constrained to a channel as shown in FIG. 3. A90-degree rotation of the magnetic field once the MicroDisk chains areconstrained in a channel will cause the MicroDisks to rotate andseparate as shown in FIG. 4. The process of steps illustrated in FIGS.2-4 comprises “magnetic arraying”.

The first step in the process, formation of chains and clusters, occursspontaneously in the presence of a magnetic field. In order to be movedinto channels clusters must be disrupted. This process is accomplishedby rotating the magnetic field. Guiding posts (discussed below in thedescription of microchannels) may be used to provide pivot points forthe rotating clusters and chains, thereby facilitating theirrearrangement. A series of properly constructed posts leads to thecreation of chains of narrow width. The chain may be wider than thewidth of a single MicroDisk.

Chains can then be moved into channels using magnet force or fluidicforce or a combination of the two. Chains will move along lines ofincreasing magnetic field strength. If the length-direction of the chain(which is substantially aligned with the preferential axis ofmagnetization of MicroDisk) aligns with or substantially aligns with themovement direction, then a smaller hydrodynamic dragging resistance isexerted on the chains, leading to a faster movement. On the other hand,it appears that, at least for individual MicroDisks, larger magneticforce is exerted on the MicroDisks if the preferential axis ofmagnetization is perpendicular or substantially perpendicular to themovement direction along which the magnetic field is increased inmagnitude. For these reasons, the chains move most efficiently when thelength-direction of the chain is at angles less than 90 degree to thedirection along which the magnetic field is increased in magnitude,typically around 45 degrees, although the chains can also move at otherdegrees. Such magnetic field gradients can be generated by largepermanent magnets or electromagnets as well as by a series of smallelectromagnets either within or adjacent to the surface of the channels.Once the MicroDisks are in the channel rotation of the magnetic field sothat it is perpendicular to the MicroDisks chain (as well as thechannel) results in the individual MicroDisks rotating to align with thefield.

Selection of optimal dimensions for the MicroDisks and channels isimportant. The amount of overlap of MicroDisks in the chains isdependent on the shape of the magnetic strips or bars within or on theMicroDisk and the thickness of the MicroDisk. In the example shown inFIG. 1, disks would be expected to overlap by 20-30% when in the chainconfiguration. By having a length to width ratio of 1.22 (90

/70

) when the MicroDisks are rotated in the channel, there is norequirement for a significant change in the relative positions of theindividual MicroDisk's center of mass within the channel due to therotation of the magnetic field. By contrast, circular MicroDisks eitherwould remain overlapped or would, as a consequence of magneticrepulsion, spread laterally through channel.

The optimal width of the channel is controlled by two factors. Thechannel must be wide enough to allow the MicroDisks to rotate, for theexample shown in FIG. 1 the diagonal of the MicroDisk is ˜114

(=√{square root over (90²+70²)}) hence this is the minimum width. Thechannel should be narrow enough to prevent two disks from forming achain when their magnetic bars or their major axis are perpendicular tothe axis of the channel when a magnetic field in the direction along thechannel with is applied. In the example shown in FIG. 1 where theMicroDisk has a dimension of 90 μ

by 70 μ

for its major surfaces, assuming an overlap of ˜30%, the length of twooverlapping MicroDisks would be ˜153 μm (=90+90−90×0.3), hence this isthe maximum width. For overlaps of 10% or 20%, the corresponding maximumwidth would be 171

m or 162 μ

. Channel height is also important since in a strong magnetic field theMicroDisks will tend to stand upright. When the constraint on themicrodevices by a magnetic field alone is sufficient to preventmicrodevices from taking such a prohibitive position, the height of themicrochannels may become irrelevant in this consideration. The arrayingprinciples discussed above and illustrated in FIGS. 2-4 are dependent onthe MicroDisks being constrained to lie flat in a plane. Consequently,the height of the channels should be less than the narrow dimension ofthe MicroDisks. A MicroDisk having angles of elevation slightly lessthan 90 degrees with respect to the bottom surface of the microchannelmay be stable if the microchannel is covered with a lid or otherwisesealed on the top. The minimum angle of elevation which still permitsstable standing of the MicroDisks is dependent on the strength of themagnetic field, the amount of magnetic material and its saturationmagnetization, as well as the weight and density of the MicroDisks andthe density of the surrounding fluid. While these values can bedetermined either empirically or through modeling, elevation angles lessthan 45 degrees would generally result in the Microdisks lying flat inthe microchannels. For these reasons, for the MicroDisk shown in FIG. 1a maximum channel height that prevents the MicroDisk standing up in thechannel is ˜50μ=(70μ×sin(45)).

The shape of the magnetic bars (or magnetic strips) within or on theMicroDisks can be tailored to direct certain types of chains andclusters to form and to alter the amount of overlap between MicroDisks.FIG. 5 shows some examples of other types of bars.

MicroDisks can be encoded in a variety of ways to make them individuallyidentifiable. The preferred encoding method is one generated during thefabrication of the MicroDisks such as 2-D bar coding or inclusion ofoptical character recognition (OCR) characters as shown in FIG. 6.

Encoded MicroDisks can be fabricated using any methods known in the art.A typical MicroDisk as shown in FIG. 1 would consist of four regions.Magnetic bars or strips are shown in light gray. Dark gray region (e.g.,made of the material Aluminum, Al) is an encoding region. Thesurrounding white edge indicates the regions that encapsulate themagnetic bars and encoding region and provide the surface formodification. This edge could be any simple material e.g., silicon,ceramic, metal, etc., though a preferred material is SiO₂. Thesedifferent regions are also located separately along the thicknessdirection. The magnetic bars and the encoding region are located in themiddle, and are encapsulated by the top and bottom layers thatcorrespond to the surrounding white edge.

The magnetic bars within or on the MicroDisks can be constructed out ofany magnetic material. Preferentially, they will be constructed out of amaterial of low magneto-restriction, low remanence, but containing ahigh saturation magnetization. For example, CoTaZr alloys meet thesecriteria. Materials of higher remanence, e.g. nickel, are compatiblewith the magnetic arraying process and may be used. The encoding layermay be constructed out of any non-magnetic material. For example,aluminum, gold, or copper could be used. Unlike encoded beads usingfluorescent labels, microfabricated bar codes such as those shown inFIG. 6 have no inherent technological limit to the number of differentcodes. FIG. 6 shows a 4-digit OCR representation, considering onlycapital and lower case letters and digits (62 characters) results inover 10⁷ possible unique representations for that type of encoding.

The ability to array allows rapid reading of encoding information on theMicroDisk without the need for complex optics with multiple orientationsand flow systems. Arrays are compatible with long-term storage andarchiving. However, unlike conventional arrays where all capturedmolecules are fixed to the same surface, in a MicroDisk array each typeof captured molecule is bound to a different surface. Consequently,individual MicroDisks can also be used for sequential methods ofanalysis. For example, following the initial screening a desired subsetof MicroDisks could be reprobed with a different detection molecule orcould be subjected to another form of analysis, e.g., sequencing or massspectroscopy. Capture on a MicroDisk in practical terms corresponds topurification of the captured molecule. Therefore, MicroDisks, whencoupled with a sorting technology, can be used to purify moieties,including proteins, DNA, cells, etc.

Another aspect of this invention is directed towards the sorting ofMicroDisks. Once inside the channel MicroDisks can be moved eitherindividually or in chains through the channel. These channels can bebranched to direct output towards different collection chambers usingmagnetic force. For example, in the case of DNA synthesis each channelcan lead to one of four tubes (A, T, C, or G). Such directional channelscould also be used to isolate specific subsets of disks for furtheranalysis (See e.g., U.S. patent application Ser. No. 09/924,428, filedAug. 7, 2001). Other sorting methods described in Section D could alsobe used for sorting of MicroDisks.

The term “magnetic bars”, in addition to rectangular shapes, includesrod-like shapes as well as slightly irregular shapes that still exhibita preferential axis of magnetization, e.g., elongated pyramidal shapes.While the examples have been confined to flat particles (MicroDisks),the microdevices of the present invention can have any shape includingspherical beads. The simplest microdevice consists of a single magneticbar that is encoded. This encoding can be created during fabricatione.g., by photolithography or it can be added after fabrication of thebar, e.g., by coupling a fluorophore.

As defined above arraying consists of displaying microdevices in anordered format such that the encoding pattern is readable. While thepreferred form of arraying is for said microdevices to be in channelswhere their preferential axis of magnetization is perpendicular to themajor axis or length axis of the microchannels, the chains of disksshown in FIG. 9 on a glass surface can already be photoanalyzed ordetected for the encoding patterns of each individual MicroDisk.Furthermore, while the preferred form of arraying is within channels (asshown in FIGS. 4 and 11), arraying can be carried out on any flatsurface e.g., a glass slide (as shown in FIG. 9). In addition, arrayscan be effectively formed in chains even if adjacent MicroDisks wouldoverlap. This can be accomplished by employing certain “accessory”MicroDisks that do not contain an encoding pattern and are transparent.By adding an appropriate excess of such transparent, “accessory”MicroDisks to the mixture of encoded MicroDisks before chain formation,the probability of two encoded MicroDisks being adjacent in the chainwill be very small. Thus, by simply forming chains of MicroDisks using amagnetic field, we can effectively achieving the arraying of the encodedMicroDisks.

While arraying is generally considered a static process, this need notbe the case. For example, particles can be moved through channels andthe encoding pattern and other information be read. The encoding patternand other information can be read by any suitable sorting instrumentse.g., FACS machines, while sorting is carried out.

In addition to enabling arraying, microdevices with a preferential axisof magnetization are able to rotate in a controlled manner within achannel in response to changes in the direction of the external appliedmagnetic field. This rotation facilitates mixing, thereby enhancingreaction kinetics and solution uniformity.

F. Examples

Protein Profiling

Encoded MicroDisks bearing a SiO₂ surface are coated using a silane toprovide activatable functional groups, e.g. coating with3-aminoproplytrimethoxysilane to provide an amine surface. Thefunctional groups are activated for coupling e.g. an amine surface isactivated using N-hydroxysulfosuccinimide and1-ethyl-3-(3-dimethylaminopropyl)carbodiimide and capture antibodies arecovalently linked to the surface through primary amino groups. Many suchencoded MicroDisks, each containing a different capture antibody, can bemade in this manner. Antibody-containing MicroDisks are then incubatedwith a sample containing antigens (or proteins) recognized by thecapture antibodies and biotinylated detection antibodies that recognizethose same antigens (or proteins). After a suitable incubation time,fluorescently labeled streptavidin is added and after furtherincubation, the MicroDisks are arrayed and subjected to analysis on anoptical reader to detect the encoding pattern and on a fluorescencereader to determine the level of bound antigen (or protein).

mRNA/cDNA Profiling

Encoded MicroDisks encapsulated in SiO₂ are modified using a silane togenerate an aldehyde surface—the preferred chemistry for linkingsynthetic oligonucleotides to a surface (see “Comparison betweendifferent strategies of covalent attachment of DNA to glass surfaces tobuild DNA microarrays” by Zammatteo et. al. Anal. Biochem., 280:143-150(2000)). This can be accomplished by coating the MicroDisks bearing an—SiO₂ surface with 3-glycidoxyproplytrimethoxysilane and hydrolyzing theresulting epoxide to a diol. The diol surface is converted to analdehyde by periodate oxidation and an amino-tagged synthetic captureoligonucleotide is covalently linked to the surface. Many such encodedMicroDisks, each containing a different capture oligonucleotides, can bemade in this manner. Oligonucleotide-containing MicroDisks are thenincubated with a sample containing fluorescently-labeled cDNAcomplementary to the capture oligonucleotide. After a suitableincubation time and washing steps, the MicroDisks are arrayed andsubjected to analysis on an optical reader to detect the encodingpattern and on a fluorescence reader to determine the level of boundantigen.

Library Synthesis

In the absence of a device or instrument that can sort individualMicroDisks, library synthesis is random. Using the split and pool methodlibraries can be synthesized directly onto the MicroDisks. After eachstep in the synthesis, the MicroDisks are arrayed and optically decodedbefore proceeding onto the next synthesis cycle. For example in the caseof DNA, after the first cycle the disks are mixed and divided into fourgroups, one group each for A, C, T, and G bases. The four groups arearrayed and optically decoded and the information is stored. The processis then repeated for each cycle. At the end of the synthesis theidentity of the oligonucleotide on each MicroDisk is known. In thismethod of random library synthesis, no two microdevices should have samephotorecognizable coding/encoding pattern, because two microdevices withsame photorecognizable coding pattern may go through different synthesiscycles and result in different synthesized entities with no method todistinguish between them. In other words, for this example, eachmicrodevice must have a unique photorecognizable coding pattern. On theother hand, in this method of random library synthesis, it is possiblefor two microdevices having different photorecognizable coding patternsto go through same synthesis cycles, resulting in their having the samesynthesized entities. The synthesized libraries can be used forscreening. Such a library-synthesis technique could also be used togenerate peptide libraries. Any library typically generated on beadscould be synthesized on MicroDisks. A very large number of suchlibraries are known to those practiced in the art of combinatorialchemistry (e.g. “Comprehensive survey of combinatorial librarysynthesis; 1999” by Dolle Journal of Combinatorial Chemistry, 2:383-433(2000)). This technique requires that each MicroDisk contain a uniquecode.

A second and more valuable method of library synthesis involves the useof a sorting step after each synthesis cycle. In this method,individually encoded MicroDisks are assigned a target sequence prior tothe initiation of library synthesis. After each step in the synthesis,each MicroDisk is directed to the appropriate reaction chamber.Procedure and the specific sequences are preassigned to individualparticles. For example, in the case of synthesizing oligonucleotides anencoded MicroDisk assigned the sequence ATCAGTCATGCG (SEQ ID NO:1) wouldgo to the A tube in the first step of synthesis then to the T tube inthe second, the C tube in the third, etc. The complete space of thelibrary is determined prior to synthesis and may correspond to a subsetof the entire sequence space available e.g., 10⁷ specific 50-residueoligonucleotides out of a sequence space of 10³⁰, or in the case ofpeptides, 10⁷ specific 20-residue peptides out of a sequence space of10²⁶. In both of these examples, it therefore is possible to generatelibraries not available by random synthetic methods (or any methods).Moreover, such techniques can be used to generate genome-specificlibraries, e.g., all 50-residue oligonucleotides or all 20-residuepeptides present in the human genome. In addition, since the encodedMicroDisks are sorted at each step it is possible to generate multiplecopies of the same library in a single synthesis because all MicroDiskscontaining the same code will be sorted together at each step in thesynthesis. For screening purposes, this means that the number of copiesof individual MicroDisks can be controlled and more importantly,libraries can be subdivided or mixed with subsets of other libraries togenerate new libraries of known sequence.

A major implementation in the synthesis of libraries involves generatinga template or scaffold that contains variable regions. Many researchersand companies (e.g. Affibody, Phylos, Ribozyme Pharmaceuticals,Somalogic) have utilized such an approach to generate syntheticantibodies, enzymes, or molecules capable of specific molecularrecognition (e.g. aptamers), enzymatic activity (e.g. ribozymes), orsignaling (e.g. by fluorescence intensity or fluorescence energytransfer). A common feature of these approaches is that they rely on theuse of enzymes (In vitro or Ex vivo) to generate secondary librariesand/or to interpret the results. For example, in the case of aptamerselection, aptamers typically are generated through the SELEX process(Systematic Evolution of Ligands by Exponential enrichments—e.g. U.S.Pat. No. 6,048,698). This involves random synthesis (though flankingregions of specific “template” sequence are required) and then screeningto obtain a subpopulation with desired binding properties. Thissubpopulation is then expanded and randomized by PCR-based methods andscreened. This iterative process of expansion and screening is continueduntil an aptamer of desired specificity and affinity has been generated.

An alternative approach in which screening and expansion are carried outusing MicroDisks offers two major advantages. The first is that allrequirements that the polymer be amplifiable by an enzymatic process areremoved. Consequently, since the polymers in each library iteration canbe generated exclusively by chemical synthesis. the polymer can comprisevirtually any type or combination of subunits, e.g. nucleotide, aminoacid, small organic molecule, sugar, protein-nucleic acid, etc. Thetremendously increased diversity of MicroDisk generated librariesenhances the likelihood of being able to produce molecules that carryout particular functions under extremes of condition, e.g. using themolecules in the libraries for capturing proteins underprotein-denaturing conditions. Such libraries can be produced usingconventional bead based synthesis, but screening and production offurther generation libraries becomes rate limiting. In conventionalbead-based synthesis, a small subset is identified by analyticalmethods, e.g. mass spectroscopy, and it is impractical to evaluate theproperties of all the members of the library. However, since theidentity of each MicroDisk is known though optical decoding, all membersof a MicroDisk library can be evaluated. For example, in a library of10¹⁰ MicroDisks, the binding efficiency of all members of the librarycan be determined and a subset of sequences can be used as startingpoints to generate the next generation library. Furthermore, in eachlibrary generation information about the measured properties of alllibrary components is retained, facilitating the use of computationalapproaches to select future generations. Such computational methodsbenefit greatly from the ability to incorporate conformationalconstraints into the library, e.g., through the use of specificcrosslinks or conformationally constrained subunits. As a result of thehuge amount of information obtained during each library screening cycle,using MicroDisk technology the conventional random approach is replacedby a guided systematic one.

The above examples are included for illustrative purposes only and arenot intended to limit the scope of the invention. Many variations tothose described above are possible. Since modifications and variationsto the examples described above will be apparent to those of skill inthis art, it is intended that this invention be limited only by thescope of the appended claims.

1. A microdevice which comprises: a) a first layer of nonmagneticmaterial; b) a photorecognizable coding pattern on said substrate; andc) a second layer comprising magnetic material, wherein said microdevicehas a longest linear dimension of not more than 500 microns and has apreferential axis of magnetization, and wherein an induced magnetizationin its absolute magnitude along the preferential axis of magnetizationof said microdevice is at least 20% more than induced magnetization ofsaid microdevice along at least one other axis.
 2. The microdevice ofclaim 1, wherein the magnetic material is patterned to comprise a bar.3. The microdevice of claim 2, wherein the photorecognizable codingpattern is lithographically patterned.
 4. The microdevice of claim 2,wherein the magnetic material comprises a CoTaZr alloy.
 5. Themicrodevice of claim 2, wherein the magnetic material comprises a cobaltalloy.
 6. The microdevice of claim 2, wherein the preferential axis ofmagnetization is substantially aligned with the major axis of themicrodevice.
 7. The microdevice of claim 1, wherein the magneticmaterial is patterned to comprise at least two bars.
 8. The microdeviceof claim 7, wherein the magnetic material comprises a cobalt alloy. 9.The microdevice of claim 1, which further comprises a binding partnerthat is capable of binding to a moiety on the surface of themicrodevice.
 10. The microdevice of claim 1 wherein the magneticmaterial is a cobalt-tantalum-zirconium (CoTaZr) alloy.
 11. Themicrodevice of claim 1, wherein an induced magnetization in its absolutemagnitude along the preferential axis of the magnetization of themicrodevice is at least 50% more than an induced magnetization of themicrodevice along at least one of any other axes.
 12. The microdevice ofclaim 1, wherein an induced magnetization in its absolute magnitudealong the preferential axis of the magnetization of the microdevice isat least 100% more than an induced magnetization of the microdevicealong at least one of any other axes.
 13. The microdevice of claim 12,wherein the magnetic material is patterned to comprise a bar.
 14. Themicrodevice of claim 12, wherein the magnetic material is patterned tocomprise at least two bars.
 15. The microdevice of claim 1, wherein aninduced magnetization in its absolute magnitude along the preferentialaxis of the magnetization of the microdevice is at least five times morethan an induced magnetization of the microdevice along at least one ofany other axes.
 16. The microdevice of claim 1, wherein the nonmagneticmaterial comprises silicon dioxide.
 17. The microdevice of claim 1,wherein the photorecognizable coding pattern is lithographicallypatterned.
 18. The microdevice of claim 1, which has a thickness of nomore than 50 microns.
 19. The microdevice of claim 1, wherein themagnetic material is a transition metal composition or an alloy thereof.20. The microdevice of claim 1, wherein the magnetic material is CoTaZralloy.
 21. The microdevice of claim 1, wherein the magnetic material isa cobalt alloy.
 22. The microdevice of claim 1, wherein the magneticmaterial is a nickel alloy.
 23. The microdevice of claim 1, wherein themagnetic material is an iron alloy.
 24. The microdevice of claim 1,wherein the photorecognizable coding pattern is fabricated ormicrofabricated on the substrate.
 25. The microdevice of claim 1,wherein the photorecognizable coding pattern comprises a 1-D or 2-Dbarcode.
 26. The microdevice of claim 1, further comprising a secondphotorecognizable coding pattern on said substrate.